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Sample records for potassium katp channel

  1. Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect.

    PubMed

    Slocinska, Malgorzata; Lubawy, Jan; Jarmuszkiewicz, Wieslawa; Rosinski, Grzegorz

    2013-11-01

    In the present study, we describe the existence of mitochondrial ATP-dependent K(+) channel (mitoKATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoKATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoKATP in insect mitochondria. MitoKATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Minoxidil opens mitochondrial KATP channels and confers cardioprotection

    PubMed Central

    Sato, Toshiaki; Li, Yulong; Saito, Tomoaki; Nakaya, Haruaki

    2003-01-01

    ATP-sensitive potassium channel in the mitochondrial inner membrane (mitoKATP channel) rather than in the sarcolemma (sarcKATP channel) appears to play an important role in cardioprotection. We examined the effect of minoxidil, a potent antihypertensive agent and hair growth stimulator, on sarcKATP and mitoKATP channels in guinea-pig ventricular myocytes. Minoxidil activated a glybenclamide-sensitive sarcKATP channel current in the whole-cell recording mode with an EC50 of 182.6 μM. Minoxidil reversibly increased the flavoprotein oxidation, an index of mitoKATP channel activity, in a concentration-dependent manner. The EC50 for mitoKATP channel activation was estimated to be 7.3 μM; this value was notably ≈25-fold lower than that for sarcKATP channel activation. Minoxidil (10 μM) significantly attenuated the ouabain-induced increase of mitochondrial Ca2+ concentration, which was measured by loading cells with rhod-2 fluorescence. Furthermore, pretreatment with minoxidil (10 μM) before 20-min no-flow ischaemia significantly improved the recovery of developed tension measured after 60 min of reperfusion in coronary perfused guinea-pig ventricular muscles. These cardioprotective effects of minoxidil were completely abolished by the mitoKATP channel blocker 5-hydroxydecanoate (500 μM). Our results indicate that minoxidil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by the selective activation of mitoKATP channels. PMID:14691056

  3. 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

  4. KATP Channels in the Cardiovascular System

    PubMed Central

    Foster, Monique N.; Coetzee, William A.

    2015-01-01

    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. PMID:26660852

  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. Copyright © 2016 the American Physiological Society.

  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. Stromal Interaction Molecule 1 (STIM1) Regulates ATP-sensitive Potassium (KATP) and Store-operated Ca(2+) Channels in MIN6 β-Cells.

    PubMed

    Leech, Colin A; Kopp, Richard F; Nelson, Heather A; Nandi, Jyotirmoy; Roe, Michael W

    2017-02-10

    Stromal interaction molecule 1 (STIM1) regulates store-operated Ca(2+) entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca(2+)-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting β-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 β-cells with RNA interference inhibits SOCE and ATP-sensitive K(+) (KATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1 Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the KATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and KATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca(2+) signaling events in β-cells.

  9. Protective effects of phosphodiesterase-1 (PDE1) and ATP sensitive potassium (KATP) channel modulators against 3-nitropropionic acid induced behavioral and biochemical toxicities in experimental Huntington׳s disease.

    PubMed

    Gupta, Surbhi; Sharma, Bhupesh

    2014-06-05

    Huntington׳s disease (HD), a devastating neurodegenerative disorder, is characterized by weight loss, impairment of motor function, cognitive dysfunction, neuropsychiatric disturbances and striatal damage. Phosphodiesterase-1 (PDE1) has been implicated in various neurological diseases. Mitochondrial potassium channels in the brain take part in neuroprotection. This study has been structured to investigate the role of vinpocetine, a selective PDE1 inhibitor as well as nicorandil, selective ATP sensitive potassium (KATP) channel opener in 3-nitropropionic acid (3-NP) induced HD symptoms in rats. Systemic administration of 3-NP significantly, reduced body weight, impaired locomotion, grip strength and impaired cognition. 3-NP elicited marked oxidative stress in the brain (enhanced malondialdehyde-MDA, reduced glutathione-GSH content, superoxide dismutase-SOD and catalase-CAT), elevated brain acetylcholinesterase activity and inflammation (myeloperoxidase-MPO), with marked nitrosative stress (nitrite/nitrate) in the brain. 3-NP has also induced mitochondrial dysfunction (impaired mitochondrial NADH dehydrogenase-complex I, succinate dehydrogenase-complex II and cytochrome oxidase-complex IV) activities in the striatum of the rat. Tetrabenazine was used as a positive control. Treatment with vinpocetine, nicorandil and tetrabenazine ameliorated 3-NP induced reduction in body weight, impaired locomotion, grip strength and impaired cognition. Treatment with these drugs reduced brain striatum oxidative (MDA, GSH, SOD and CAT) and nitrosative (nitrite/nitrate) stress, acetylcholinesterase activity, inflammation and mitochondrial dysfunctions. These results indicate that vinpocetine, a selective PDE1 inhibitor and nicorandil, a KATP channel opener have attenuated 3-NP induced experimental HD. Hence, pharmacological modulation of PDE1 as well as KATP channels may be considered as potential research targets for mitigation of HD. Copyright © 2014 Elsevier B.V. All rights

  10. Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels.

    PubMed

    Kim, Do Young; Abdelwahab, Mohammed G; Lee, Soo Han; O'Neill, Derek; Thompson, Roger J; Duff, Henry J; Sullivan, Patrick G; Rho, Jong M

    2015-01-01

    Dietary and metabolic therapies are increasingly being considered for a variety of neurological disorders, based in part on growing evidence for the neuroprotective properties of the ketogenic diet (KD) and ketones. Earlier, we demonstrated that ketones afford hippocampal synaptic protection against exogenous oxidative stress, but the mechanisms underlying these actions remain unclear. Recent studies have shown that ketones may modulate neuronal firing through interactions with ATP-sensitive potassium (KATP) channels. Here, we used a combination of electrophysiological, pharmacological, and biochemical assays to determine whether hippocampal synaptic protection by ketones is a consequence of KATP channel activation. Ketones dose-dependently reversed oxidative impairment of hippocampal synaptic integrity, neuronal viability, and bioenergetic capacity, and this action was mirrored by the KATP channel activator diazoxide. Inhibition of KATP channels reversed ketone-evoked hippocampal protection, and genetic ablation of the inwardly rectifying K+ channel subunit Kir6.2, a critical component of KATP channels, partially negated the synaptic protection afforded by ketones. This partial protection was completely reversed by co-application of the KATP blocker, 5-hydoxydecanoate (5HD). We conclude that, under conditions of oxidative injury, ketones induce synaptic protection in part through activation of KATP channels.

  11. Ketones Prevent Oxidative Impairment of Hippocampal Synaptic Integrity through KATP Channels

    PubMed Central

    Kim, Do Young; Abdelwahab, Mohammed G.; Lee, Soo Han; O’Neill, Derek; Thompson, Roger J.; Duff, Henry J.; Sullivan, Patrick G.; Rho, Jong M.

    2015-01-01

    Dietary and metabolic therapies are increasingly being considered for a variety of neurological disorders, based in part on growing evidence for the neuroprotective properties of the ketogenic diet (KD) and ketones. Earlier, we demonstrated that ketones afford hippocampal synaptic protection against exogenous oxidative stress, but the mechanisms underlying these actions remain unclear. Recent studies have shown that ketones may modulate neuronal firing through interactions with ATP-sensitive potassium (KATP) channels. Here, we used a combination of electrophysiological, pharmacological, and biochemical assays to determine whether hippocampal synaptic protection by ketones is a consequence of KATP channel activation. Ketones dose-dependently reversed oxidative impairment of hippocampal synaptic integrity, neuronal viability, and bioenergetic capacity, and this action was mirrored by the KATP channel activator diazoxide. Inhibition of KATP channels reversed ketone-evoked hippocampal protection, and genetic ablation of the inwardly rectifying K+ channel subunit Kir6.2, a critical component of KATP channels, partially negated the synaptic protection afforded by ketones. This partial protection was completely reversed by co-application of the KATP blocker, 5-hydoxydecanoate (5HD). We conclude that, under conditions of oxidative injury, ketones induce synaptic protection in part through activation of KATP channels. PMID:25848768

  12. Direct interaction of Na-azide with the KATP channel.

    PubMed

    Trapp, S; Ashcroft, F M

    2000-11-01

    1. The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type. 2. In inside-out patches from oocytes expressing Kir6.2 delta C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose. 3. Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2 delta C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+. 4. Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR. 5. We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit. The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration.

  13. KATP channels process nucleotide signals in muscle thermogenic response

    PubMed Central

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

    2014-01-01

    Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K+ (KATP) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, KATP channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how KATP 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 KATP 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 KATP channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal KATP 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 KATP channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of KATP channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders. PMID:20925594

  14. Trypsin and alpha-chymotrypsin treatment abolishes glibenclamide sensitivity of KATP channels in rat ventricular myocytes.

    PubMed

    Nichols, C G; Lopatin, A N

    1993-03-01

    Cytoplasmic trypsin-treatment of voltage-sensitive potassium channels has been shown to cleave domains of the channel responsible for inactivation of the channel. Trypsin has also been reported to remove slow, irreversible inactivation, or run-down in ATP-sensitive potassium (KATP) channels. Cytoplasmic treatment of rat ventricular KATP channels with either crude, or pure trypsin (1-2 mg/ml) failed to prevent a slow run-down of channel activity. However, trypsin (porcine pancreatic type IX, or type II (Sigma Chem. Co.), or alpha-chymotrypsin (Sigma Chem. Co.) rapidly and irreversibly removed, or substantiallly decreased glibenclamide and tolbutamide-sensitivity of the channels without removing sensitivity to ATP. We conclude that glibenclamide must bind to either a separate protein, or to a separate domain on the channel in order to effect channel inhibition, and this domain is functionally disconnected from the channel by trypsin-, or alpha-chymotrypsin treatment.

  15. Structure and function of the low conductance KATP channel, ROMK.

    PubMed

    Hebert, S C; Wang, W H

    1997-06-27

    The renal ATP-sensitive low-conductance K+ channel (KATP) plays an important role in K+ recycling in the thick ascending limb and in K+ secretion in the collecting duct. The low-conductance KATP is stimulated by cAMP-dependent protein kinase A and inhibited by protein kinase C, arachidonic acid, acidic pH and sulfonylurea agents. We reviewed the progress concerning the properties of the recently cloned inward-rectifying K+ channel (ROMK or KirI) and compared their regulatory mechanisms with the native low-conductance KATP. The results are important to gain insight into molecular mechanisms by which ROMK channels are regulated.

  16. Neuroprotective effect of KR-31378 via KATP channel opening against ischemic insult.

    PubMed

    Won, Ran; Lim, Jong-Yoon; Lee, Sang-Yeon; Park, Ji-Ho; Sohn, Nak-Won

    2004-08-01

    The opening of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel has been proposed as a therapeutic approach for ischemia. Here we examined the opening effect of KR-31378 on the KATP channel using patch clamp recording in neuroblastoma 2a (N2a) cells and investigated the neuroprotective effect of KR-31378 in organotypic hippocampal slice cultures exposed to oxygen/glucose deprivation. The treatment with KR-31378 (10 microM) to N2a cells seemed to induce KATP channel opening in a dose dependent manner. The opening effect of KR-31378 was more significant than that of other known KATP channel openers. Pretreatment with KR-31378 (10 microM) showed a neuroprotective effect in both CA1 and CA3 regions and its effect was attenuated by glibenclamide in a dose dependent manner in both areas. This remarkable neuroprotective effect of KR-31378 seemed to be mediated by the opening of the KATP channel. These results suggest that KR-31378 could be a possible neuroprotective agent against cerebral ischemia.

  17. KATP channels are common mediators of ischemic and calcium preconditioning in rabbits.

    PubMed

    Kouchi, I; Murakami, T; Nawada, R; Akao, M; Sasayama, S

    1998-04-01

    Calcium preconditioning (CPC), like ischemic preconditioning (IPC), reduces myocardial infarct size in dogs and rats. ATP-sensitive potassium (KATP) channels induce cardioprotection of IPC in these animals. To determine whether KATP channels mediate both IPC and CPC, pentobarbital sodium-anesthetized rabbits received 30 min of coronary artery occlusion followed by 180 min of reperfusion. IPC was elicited by 5 min of occlusion and 10 min of reperfusion, and CPC was elicited by two cycles of 5 min of calcium infusion with an interval period of 15 min. Infarct size expressed as a percentage of the area at risk was 38 +/- 3% (mean +/- SE) in controls. IPC, CPC, and pretreatment with a KATP channel opener, cromakalim, all reduced infarct size to 13 +/- 2, 17 +/- 2, and 12 +/- 3%, respectively (P < 0.01 vs. controls). Glibenclamide, a KATP channel blocker administered 45 min (but not 20 min) before sustained ischemia, attenuated the effects of IPC and CPC (31 +/- 4 and 41 +/- 6%, respectively). Thus KATP channel activation appears to contribute to these two types of cardioprotection in rabbits.

  18. Rotenone induces KATP channel opening in PC12 cells in association with the expression of tyrosine hydroxylase.

    PubMed

    Bai, Qunhua; He, Junlin; Qiu, Jingfu; Wang, Yang; Wang, Shibo; Xiu, Yun; Yu, Chao

    2012-10-01

    The activation of ATP-sensitive potassium (KATP) channels in PC12 cells play a pivotal role in protection against the neurotoxic effect of rotenone. However, it remains unclear why rotenone seems to preferentially affect activation of KATP channels and if this could affect its physiological activity. In this study, we sought to determine how the different energy states caused by various doses of rotenone affect the KATP opening state and whether the KATP opening state influences the expression of tyrosine hydroxylase (TH) which is related with DA synthesis. With patch clamp technology, results showed that treatment of PC12 cells with rotenone (0.2-1 µg/ml) for 15 min can cause KATP channel opening with significantly increased intracellular ROS production. Treatment with rotenone (2-16 ng/ml) for 24 h also caused the channels to open with gently increased ROS. In order to study if the rather long-term action on KATP channel opening states could affect the specified function of PC12 cells, the KATP channel opener pinacidil and the inhibitor glibenclamide were used to treat cells for 24 h, and the expression of TH was detected. Our results showed that treatment of PC12 cells with glibenclamide for 24 h can notably promote TH expression and can also enhance the expression of TH which were reduced by rotenone. These data indicate that the energy states in PC12 induced by various doses of rotenone could significantly influence the opening states of KATP channels. However long-term energy stress may raise the opening rate and opening sensitivity of this channel. In addition, our results demonstrate for the first time that activation of plasma membrane KATP channels induced by rotenone inhibits TH expression which influences DA synthesis in PC12 cells.

  19. 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

  20. Muscle KATP Channels: Recent Insights to Energy Sensing and Myoprotection

    PubMed Central

    Flagg, Thomas P.; Enkvetchakul, Decha; Koster, Joseph C.; Nichols, Colin G.

    2011-01-01

    ATP-sensitive (KATP) channels are present in the surface and internal membranes of cardiac, skeletal and smooth muscle cell, and provide a unique feedback between muscle cell metabolism and electrical activity. In so doing, they can play an important role in the control of contractility, particularly when cellular energetics are compromised, protecting the tissue against calcium overload and fiber damage, but the cost of this protection may be enhanced arrhythmic activity. Generated as complexes of Kir6.1 or Kir6.2 pore-forming subunits with regulatory sulfonylurea receptor subunits, SUR1 or SUR2, the differential assembly of KATP channels in different tissues gives rise to tissue-specific physiological and pharmacological regulation, and hence to the tissue-specific pharmacological control of contractility. The last ten years have provided insights to the regulation and role of muscle KATP channels, in large part driven by studies of mice in which the protein determinants of channel activity have been deleted or modified. As yet, few human diseases have been correlated with altered muscle KATP activity, but genetically modified animals give important insights to likely pathological roles of aberrant channel activity in different muscle types. PMID:20664073

  1. Chronic exposure to KATP channel openers results in attenuated glucose sensing in hypothalamic GT1-7 neurons.

    PubMed

    Haythorne, Elizabeth; Hamilton, D Lee; Findlay, John A; Beall, Craig; McCrimmon, Rory J; Ashford, Michael L J

    2016-12-01

    Individuals with Type 1 diabetes (T1D) are often exposed to recurrent episodes of hypoglycaemia. This reduces hormonal and behavioural responses that normally counteract low glucose in order to maintain glucose homeostasis, with altered responsiveness of glucose sensing hypothalamic neurons implicated. Although the molecular mechanisms are unknown, pharmacological studies implicate hypothalamic ATP-sensitive potassium channel (KATP) activity, with KATP openers (KCOs) amplifying, through cell hyperpolarization, the response to hypoglycaemia. Although initial findings, using acute hypothalamic KCO delivery, in rats were promising, chronic exposure to the KCO NN414 worsened the responses to subsequent hypoglycaemic challenge. To investigate this further we used GT1-7 cells to explore how NN414 affected glucose-sensing behaviour, the metabolic response of cells to hypoglycaemia and KATP activity. GT1-7 cells exposed to 3 or 24 h NN414 exhibited an attenuated hyperpolarization to subsequent hypoglycaemic challenge or NN414, which correlated with diminished KATP activity. The reduced sensitivity to hypoglycaemia was apparent 24 h after NN414 removal, even though intrinsic KATP activity recovered. The NN414-modified glucose responsiveness was not associated with adaptations in glucose uptake, metabolism or oxidation. KATP inactivation by NN414 was prevented by the concurrent presence of tolbutamide, which maintains KATP closure. Single channel recordings indicate that NN414 alters KATP intrinsic gating inducing a stable closed or inactivated state. These data indicate that exposure of hypothalamic glucose sensing cells to chronic NN414 drives a sustained conformational change to KATP, probably by binding to SUR1, that results in loss of channel sensitivity to intrinsic metabolic factors such as MgADP and small molecule agonists.

  2. 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.

  3. Stimulation of neuronal KATP channels by cGMP-dependent protein kinase: involvement of ROS and 5-hydroxydecanoate-sensitive factors in signal transduction

    PubMed Central

    Chai, Yongping

    2010-01-01

    The ATP-sensitive potassium (KATP) channel couples intracellular metabolic state to membrane excitability. Recently, we demonstrated that neuronal KATP channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal KATP channels. By performing single-channel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type KATP) channel currents by PKG activation in cell-attached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial KATP channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H2O2)-decomposing enzyme. These reagents also ablated NO-induced KATP channel stimulation and prevented the shifts in the single-channel open- and closed-time distributions resulting from PKG activation and NO induction. Bath application of H2O2 reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H2O2 was able to enhance the function of KATP channels in the presence of Ca2+ chelators and calmodulin antagonists, whereas the stimulatory effect of H2O2 was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal KATP channels by modulating intrinsic channel gating via a 5-HD-sensitive factor(s)/ROS/Ca2+/calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal KATP channels. PMID:20053925

  4. Expression and function of ATP-dependent potassium channels in zebrafish islet β-cells

    PubMed Central

    Emfinger, Christopher H.; Welscher, Alecia; Yan, Zihan; Wang, Yixi; Conway, Hannah; Moss, Jennifer B.; Moss, Larry G.; Remedi, Maria S.

    2017-01-01

    ATP-sensitive potassium channels (KATP channels) are critical nutrient sensors in many mammalian tissues. In the pancreas, KATP channels are essential for coupling glucose metabolism to insulin secretion. While orthologous genes for many components of metabolism–secretion coupling in mammals are present in lower vertebrates, their expression, functionality and ultimate impact on body glucose homeostasis are unclear. In this paper, we demonstrate that zebrafish islet β-cells express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. We further show that pharmacological activation of native zebrafish KATP using diazoxide, a specific KATP channel opener, is sufficient to disturb glucose tolerance in adult zebrafish. That β-cell KATP channel expression and function are conserved between zebrafish and mammals illustrates the evolutionary conservation of islet metabolic sensing from fish to humans, and lends relevance to the use of zebrafish to model islet glucose sensing and diseases of membrane excitability such as neonatal diabetes. PMID:28386438

  5. Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia

    PubMed Central

    Sun, Hong-shuo; Feng, Zhong-ping

    2013-01-01

    ATP-sensitive potassium (KATP) channels are weak, inward rectifiers that couple metabolic status to cell membrane electrical activity, thus modulating many cellular functions. An increase in the ADP/ATP ratio opens KATP channels, leading to membrane hyperpolarization. KATP channels are ubiquitously expressed in neurons located in different regions of the brain, including the hippocampus and cortex. Brief hypoxia triggers membrane hyperpolarization in these central neurons. In vivo animal studies confirmed that knocking out the Kir6.2 subunit of the KATP channels increases ischemic infarction, and overexpression of the Kir6.2 subunit reduces neuronal injury from ischemic insults. These findings provide the basis for a practical strategy whereby activation of endogenous KATP channels reduces cellular damage resulting from cerebral ischemic stroke. KATP channel modulators may prove to be clinically useful as part of a combination therapy for stroke management in the future. PMID:23123646

  6. K(ATP) channel block prevents proteasome inhibitor-induced apoptosis in differentiated PC12 cells.

    PubMed

    Nam, Yoon Jeong; Lee, Da Hee; Lee, Min Sung; Lee, Chung Soo

    2015-10-05

    Dysfunction of the proteasome system has been suggested to be implicated in neuronal degeneration. Modulation of KATP channels appears to affect the viability of neuronal cells exposed to toxic insults. However, the effect of KATP channel blockers on the neuronal cell death mediated by proteasome inhibition has not been studied. The present study investigated the effect of KATP channel blockers on proteasome inhibitor-induced apoptosis in differentiated PC12 cells and SH-SY5Y cells. 5-Hydroxydecanoate (a selective KATP channel blocker) and glibenclamide (a cell surface and mitochondrial KATP channel inhibitor) reduced the proteasome inhibitor-induced apoptosis. Addition of the KATP channel blockers attenuated the proteasome inhibitor-induced changes in the levels of apoptosis-related proteins, the loss of the mitochondrial transmembrane potential, the increase in the formation of reactive oxygen species and the depletion of glutathione in both cell lines. The results show that KATP channel blockers may attenuate proteasome inhibitor-induced apoptosis in PC12 cells by suppressing activation of the mitochondrial pathway and of the caspase-8- and Bid-dependent pathways. The preventive effect appears to be associated with the inhibition of the formation of reactive oxygen species and the depletion of glutathione. KATP channel blockade appears to prevent proteasome inhibition-induced neuronal cell death.

  7. 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

  8. Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: possible role in glucose sensing.

    PubMed

    Rufino, Ana T; Rosa, Susana C; Judas, Fernando; Mobasheri, Ali; Lopes, M Celeste; Mendes, Alexandrina F

    2013-08-01

    ATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes.

  9. 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.

  10. Effects of ATP-sensitive potassium channel opener on potassium transport and alveolar fluid clearance in the resected human lung.

    PubMed

    Sakuma, T; Takahashi, K; Ohya, N; Nakada, T; Matthay, M A

    1998-07-01

    Since the effect of an ATP-sensitive potassium channel (KATP channel) opener on the function of alveolar epithelial cells is unknown, the effect of YM934, a newly synthesized KATP channel opener, on potassium influx into the alveolar spaces and alveolar fluid clearance was determined in the resected human lung. An isosmolar albumin solution with a low potassium concentration was instilled into the distal airspaces of resected human lungs. Alveolar fluid clearance was measured by the progressive increase in alveolar protein concentration. Net potassium transport was measured by the change in potassium concentration and alveolar fluid volume. YM934 (10(-4) M) increased net influx of potassium by 140% into the alveolar spaces and also increased alveolar fluid clearance by 60% in the experiments with a potassium concentration of 0.3 mEq/1. Glibenclamide (10(-4) M), a KATP channel blocker, inhibited the YM934-increased influx of potassium transport and the increase in alveolar fluid clearance. Also amiloride (10(-5) M), an inhibitors of apical sodium uptake, blocked the YM934 stimulated increase in net alveolar fluid clearance. These results indicate that a KATP channel opener can effect potassium transport and net vectorial fluid movement across the human alveolar epithelium.

  11. Acute Simvastatin Inhibits KATP Channels of Porcine Coronary Artery Myocytes

    PubMed Central

    Zhang, Qian; Li, Rachel Wai Sum; Kong, Siu Kai; Ngai, Sai Ming; Wan, Song; Ho, Ho Pui; Lee, Simon Ming Yuen; Hoi, Maggie Pui Man; Chan, Shun Wan; Leung, George Pak Heng; Kwan, Yiu Wa

    2013-01-01

    Background Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular KATP channel gatings are unknown. Methods Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca2+]i, [ATP]i and [glucose]o uptake measurements. Results The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell KATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [3H]-2-deoxy-glucose uptake and an increase in [ATP]i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr172 and p-PP2A-Tyr307 expression was observed. The enhanced p-AMPKα-Thr172 expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr307 expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]o-free or [Na+]o-free conditions. Conclusions Simvastatin causes ryanodine-sensitive Ca2+ release which is important for AMPKα-Thr172 phosphorylation via Ca2+/CaMK II. AMPKα-Thr172 phosphorylation causes [glucose]o uptake (and an [ATP]i increase), closure of KATP channels, and phosphorylation of AMPKα-Thr172 and PP2A-Tyr307 resulted. Phosphorylation of PP2A-Tyr307 occurs at a site downstream of AMPKα-Thr172 phosphorylation. PMID:23799098

  12. Allicin relaxes isolated mesenteric arteries through activation of PKA-KATP channel in rat.

    PubMed

    Li, Shuzhen; Chen, Shuo; Yang, Weiwei; Liao, Lin; Li, Shanshan; Li, Jiali; Zheng, Yaqin; Zhu, Daling

    2017-02-01

    Allicin is a natural effective organosulfur compound isolated from garlic, which possesses many beneficial properties, such as antibacterial, anti-inflammatory, antimicrobial, hypotensive and hypolipidemic. In the present study, we investigated the effects and the underlying mechanisms of allicin on isolated mesenteric arteries (MAs). We examined MAs relaxation induced by allicin on rat-isolated mesenteric artery (MA) rings, the KATP channels with patch, and the expression of Kir6.1 and SUR2B with western blotting and NO production with Diaminofluorescein-FM diacetate (DAF-FMDA) in rat mesenteric artery smooth muscle cells (MASMCs). The results showed that allicin elicited the dose-dependent vasorelaxation effect with phenylephrine (PE) precontracted rat MA rings. The vasorelaxation effect was endothelium and NO independent but could be diminished by inhibition of PKA and KATP channels in the vascular smooth muscle. Allicin activated KATP channels in rat MASMCs, and the activation of KATP channels was inhibited by the inhibitors of PKA and KATP channels. But allicin had no effect on the expression of KATP subtypes Kir6.1 and SUR2B. These observations suggest that allicin exerts vasorelaxation effect through activation of PKA-KATP(-)signaling pathway.

  13. The KATP channel is a molecular sensor of atrophy in skeletal muscle

    PubMed Central

    Tricarico, Domenico; Mele, Antonietta; Camerino, Giulia Maria; Bottinelli, Roberto; Brocca, Lorenza; Frigeri, Antonio; Svelto, Maria; George, Alfred L; Camerino, Diana Conte

    2010-01-01

    The involvement of ATP-sensitive K+ (KATP) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of KATP channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in KATP currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and KATP currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced KATP currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the KATP channel blocker glibenclamide (10−6m) reduced the KATP currents with atrophy and these effects were prevented by the KATP channel opener diazoxide (10−4m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the KATP channel as a molecular sensor of atrophy. PMID:20064856

  14. Molecular mechanism for H(2)S-induced activation of K(ATP) channels.

    PubMed

    Jiang, Bo; Tang, Guanghua; Cao, Kun; Wu, Lingyun; Wang, Rui

    2010-05-15

    Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

  15. Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.

    PubMed

    Chen, Pei-Chun; Olson, Erik M; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M; Thomas, David Y; Shyng, Show-Ling

    2013-07-19

    ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

  16. Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism*

    PubMed Central

    Chen, Pei-Chun; Olson, Erik M.; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M.; Thomas, David Y.; Shyng, Show-Ling

    2013-01-01

    ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients. PMID:23744072

  17. H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal K(ATP) channel.

    PubMed

    Fitzgerald, Robert; DeSantiago, Breann; Lee, Danielle Y; Yang, Guangdong; Kim, Jae Yeon; Foster, D Brian; Chan-Li, Yee; Horton, Maureen R; Panettieri, Reynold A; Wang, Rui; An, Steven S

    2014-03-28

    Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)-the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1-10mM). GYY4137 slowly released appreciable levels of H2S in the range of 10-275 μM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

  18. Expression of ATP-sensitive potassium channels in human pregnant myometrium

    PubMed Central

    2011-01-01

    Background Potassium channels play critical roles in the regulation of cell membrane potential, which is central to the excitability of myometrium. The ATP-sensitive potassium (KATP) channel is one of the most abundant potassium channels in myometrium. The objectives of this study were to investigate the protein expression of KATP channel in human myometrium and determine the levels of KATP channel in lower and upper segmental myometrium before and after onset of labour. Methods Both lower segmental (LS) and upper segmental (US) myometrial biopsies were collected at cesarean section from pregnant women not-in-labour (TNL) or in-labour (TL) at term. Protein expression level and cellular localization of four KATP channel subunits in US and LS myometrium were determined by Western blot analysis and immunohistochemistry, respectively. The contractile activity of myometrial strip was measured under isometric conditions. Results Four KATP channel subunits, namely Kir6.1, Kir6.2, SUR1 and SUR2B were identified in pregnant myometrium. While found in vascular myocytes, these subunits appear to be preferentially expressed in myometrial myocytes. Diazoxide, a KATP channel opener, inhibited the spontaneous contractility of pregnant myometrium, suggesting that the KATP channels are functional in human pregnant myometrium. Diazoxide was less potent in TL strips than that in TNL strips. Interestingly, expression of SUR1 was greater in TL than TNL tissues, although no differences were found for SUR2B in these two tissues. For both lower and upper segmental myometrium, Kir6.1 and Kir6.2 were less in TL compared with TNL tissues. Conclusions Functional KATP channels are expressed in human pregnant myometrium. Down-regulation of Kir6.1 and Kir6.2 expression in myometrium may contribute to the enhanced uterine contractility associated with the onset of labour. PMID:21418633

  19. Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

    PubMed Central

    Yan, Fei-Fei; Pratt, Emily B.; Chen, Pei-Chun; Wang, Fang; Skach, William R.; David, Larry L.

    2010-01-01

    The pancreatic β-cell ATP-sensitive potassium (KATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. KATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing KATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with KATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell KATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type KATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric KATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane. PMID:20427569

  20. Cardiac specific ATP-sensitive K+ channel (KATP) overexpression results in embryonic lethality.

    PubMed

    Toib, Amir; Zhang, Hai Xia; Broekelmann, Thomas J; Hyrc, Krzysztof L; Guo, Qiusha; Chen, Feng; Remedi, Maria S; Nichols, Colin G

    2012-09-01

    Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.

  1. ATP-dependent potassium channels and type 2 diabetes mellitus.

    PubMed

    Bonfanti, Dianne Heloisa; Alcazar, Larissa Pontes; Arakaki, Priscila Akemi; Martins, Laysa Toschi; Agustini, Bruna Carla; de Moraes Rego, Fabiane Gomes; Frigeri, Henrique Ravanhol

    2015-05-01

    Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

  2. Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

    PubMed Central

    Martin, Gregory M.; Chen, Pei-Chun; Devaraneni, Prasanna; Shyng, Show-Ling

    2013-01-01

    ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed. PMID:24399968

  3. Nandrolone decanoate negatively reverses the beneficial effects of exercise on cardiac muscle via sarcolemmal, but not mitochondrial K(ATP) channel.

    PubMed

    Bayat, Gholamreza; Javan, Mohammad; Safari, Fatemeh; Khalili, Azadeh; Shokri, Saeed; Goudarzvand, Mahdi; Salimi, Mehdi; Hajizadeh, Sohrab

    2016-03-01

    ATP-sensitive potassium channels are supposed to have a substantial role in improvement of cardiac performance. This study was performed to evaluate whether nandrolone decanoate (ND) and (or) exercise training could affect the expression of cardiac K(ATP) channel subunits. Thirty-five male albino Wistar rats were randomly divided into 5 groups, including sedentary control (SC), sedentary vehicle (SV), sedentary ND (SND), exercise control (EC), and exercise and ND (E+ND). Exercise training was performed on a treadmill 5 times per week. ND was injected (10 mg/kg/week, i.m.) to the rats in the SND and E+ND groups. Following cardiac isolation, the expression of both sarcolemmal and mitochondrial subunits of K(ATP) channel was measured using Western blot method. The expression of sarcolemmal, but not mitochondrial, subunits of K(ATP) channel (Kir6.2 and SUR2) of EC group was significantly higher compared with SC group while ND administration (SND group) did not show any change in their expression. In the E+ND group, ND administration led to decrease of the over-expression of sarcolemmal Kir6.2 and SUR2 which was previously induced by exercise. There was no significant association between the mitochondrial expression of either Kir6.2 or SUR2 proteins and administration of ND or exercise. Supra-physiological dosage of ND negatively reverses the effects of exercise on the cardiac muscle expression of sarcolemmal, but not mitochondrial, K(ATP) channel subunits.

  4. The KATP channel is a molecular sensor of atrophy in skeletal muscle.

    PubMed

    Tricarico, Domenico; Mele, Antonietta; Camerino, Giulia Maria; Bottinelli, Roberto; Brocca, Lorenza; Frigeri, Antonio; Svelto, Maria; George, Alfred L; Camerino, Diana Conte

    2010-03-01

    The involvement of ATP-sensitive K(+) (K(ATP)) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of K(ATP) channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in K(ATP) currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and K(ATP) currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced K(ATP) currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the K(ATP) channel blocker glibenclamide (10(6)m) reduced the K(ATP) currents with atrophy and these effects were prevented by the K(ATP) channel opener diazoxide (10(4)m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the K(ATP) channel as a molecular sensor of atrophy.

  5. Autocrine insulin increases plasma membrane K(ATP) channel via PI3K-VAMP2 pathway in MIN6 cells.

    PubMed

    Xu, Shanhua; Kim, Ji-Hee; Hwang, Kyu-Hee; Das, Ranjan; Quan, Xianglan; Nguyen, Tuyet Thi; Kim, Soo-Jin; Cha, Seung-Kuy; Park, Kyu-Sang

    2015-12-25

    Regulation of ATP-sensitive inwardly rectifying potassium (KATP) channel plays a critical role in metabolism-secretion coupling of pancreatic β-cells. Released insulin from β-cells inhibits insulin and glucagon secretion with autocrine and paracrine modes. However, molecular mechanism by which insulin inhibits hormone secretion remains elusive. Here, we investigated the effect of autocrine insulin on surface abundance of KATP channel in mouse clonal β-cell line, MIN6. High glucose increased plasmalemmal sulfonylurea receptor 1 (SUR1), a component of KATP channel as well as exogenous insulin treatment. SUR1 trafficking by high glucose or insulin was blocked by inhibition of phosphoinositide 3-kinase (PI3K) with wortmannin. Pretreatment with brefeldin A or silencing of vesicle-associated membrane protein 2 (VAMP2) abolished insulin-mediated upregulation of surface SUR1. Functionally, glucose-stimulated cytosolic Ca(2+) ([Ca(2+)]i) increase was blunted by insulin or diazoxide, a KATP channel opener. Insulin-induced suppression of [Ca(2+)]i oscillation was prevented by an insulin receptor blocker. These results provide a novel molecular mechanism for autocrine negative feedback regulation of insulin secretion.

  6. BAD-Dependent Regulation of Fuel Metabolism and KATP Channel Activity Confers Resistance to Epileptic Seizures

    PubMed Central

    Giménez-Cassina, Alfredo; Martínez-François, Juan Ramón; Fisher, Jill K.; Szlyk, Benjamin; Polak, Klaudia; Wiwczar, Jessica; Tanner, Geoffrey R.; Lutas, Andrew; Yellen, Gary; Danial, Nika N.

    2012-01-01

    Summary Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phospho-regulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive KATP channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the KATP channel, implicating the BAD-KATP axis in metabolic control of neuronal excitation and seizure responses. PMID:22632729

  7. Nitric oxide activates ATP-sensitive potassium channels in mammalian sensory neurons: action by direct S-nitrosylation

    PubMed Central

    Kawano, Takashi; Zoga, Vasiliki; Kimura, Masakazu; Liang, Mei-Ying; Wu, Hsiang-En; Gemes, Geza; McCallum, J Bruce; Kwok, Wai-Meng; Hogan, Quinn H; Sarantopoulos, Constantine D

    2009-01-01

    Background ATP-sensitive potassium (KATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons. Results Cell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i. This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit. Conclusion NO activates KATP channels in

  8. 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

  9. Role of KATP channels in the maintenance of ventricular fibrillation in cardiomyopathic human hearts.

    PubMed

    Farid, Talha A; Nair, Krishnakumar; Massé, Stephéne; Azam, Mohammed Ali; Maguy, Ange; Lai, Patrick F H; Umapathy, Karthikeyan; Dorian, Paul; Chauhan, Vijay; Varró, András; Al-Hesayen, Abdul; Waxman, Menashe; Nattel, Stanley; Nanthakumar, Kumaraswamy

    2011-11-11

    Ventricular fibrillation (VF) leads to global ischemia. The modulation of ischemia-dependent pathways may alter the electrophysiological evolution of VF. We addressed the hypotheses that there is regional disease-related expression of K(ATP) channels in human cardiomyopathic hearts and that K(ATP) channel blockade promotes spontaneous VF termination by attenuating spatiotemporal dispersion of refractoriness. In a human Langendorff model, electric mapping of 6 control and 9 treatment (10 μmol/L glibenclamide) isolated cardiomyopathic hearts was performed. Spontaneous defibrillation was studied and mean VF cycle length was compared regionally at VF onset and after 180 seconds between control and treatment groups. K(ATP) subunit gene expression was compared between LV endocardium versus epicardium in myopathic hearts. Spontaneous VF termination occurred in 1 of 6 control hearts and 7 of 8 glibenclamide-treated hearts (P=0.026). After 180 seconds of ischemia, a transmural dispersion in VF cycle length was observed between epicardium and endocardium (P=0.001), which was attenuated by glibenclamide. There was greater gene expression of all K(ATP) subunit on the endocardium compared with the epicardium (P<0.02). In an ischemic rat heart model, transmural dispersion of refractoriness (ΔERP(Transmural)=ERP(Epicardium)-ERP(Endocardium)) was verified with pacing protocols. ΔERP(Transmural) in control was 5 ± 2 ms and increased to 36 ± 5 ms with ischemia. This effect was greatly attenuated by glibenclamide (ΔERP(Transmural) for glibenclamide+ischemia=4.9 ± 4 ms, P=0.019 versus control ischemia). K(ATP) channel subunit gene expression is heterogeneously altered in the cardiomyopathic human heart. Blockade of K(ATP) channels promotes spontaneous defibrillation in cardiomyopathic human hearts by attenuating the ischemia-dependent spatiotemporal heterogeneity of refractoriness during early VF.

  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 currents from channels formed by Kir6 and a modified cardiac mitochondrial SUR2 variant

    PubMed Central

    Aggarwal, Nitin T; Shi, Nian-Qing; Makielski, Jonathan C

    2013-01-01

    Cardiac ATP-sensitive potassium channels (KATP) are found in both the sarcoplasmic reticulum (sarcKATP) and the inner membrane of mitochondria (mitoKATP). SarcKATP are composed of a pore containing subunit Kir6.2 and a regulatory sulfonylurea receptor subunit (SUR2), but the composition of mitoKATP remains unclear. An unusual intra-exonic splice variant of SUR2 (SUR2A-55) was previously identified in mitochondria of mammalian heart and brain, and by analogy with sarcKATP we proposed SUR2A-55 as a candidate regulatory subunit of mitoKATP. Although SUR2A-55 lacks the first nucleotide binding domain (NBD) and 2 transmembrane domains (TMD), it has a hybrid TMD and retains the second NBD. It resembles a hemi-ABC transporter suggesting it could multimerize to function as a regulatory subunit. A putative mitochondrial targeting signal in the N-terminal domain of SUR2A-55 was removed by truncation and when co-expressed with Kir6.1 and Kir6.2 it targeted to the plasma membrane and yielded KATP currents. Single channel conductance, mean open time, and burst open time of SUR2A-55 based KATP was similar to the full-length SUR2A based KATP. However, the SUR2A-55 KATP were 70-fold less sensitive to block by ATP, and twice as resistant to intracellular Ca2+ inhibition compared with the SUR2A KATP, and were markedly insensitive to KATP drugs, pinacidil, diazoxide, and glybenclamide. These results suggest that the SUR2A-55 based channels would tend to be open under physiological conditions and in ischemia, and could account for cardiac and mitochondrial phenotypes protective for ischemia. PMID:24037327

  12. The antiallodynic action target of intrathecal gabapentin: Ca2+ channels, KATP channels or N-methyl-d-aspartic acid receptors?

    PubMed

    Cheng, Jen-Kun; Chen, Chien-Chuan; Yang, Jia-Rung; Chiou, Lih-Chu

    2006-01-01

    Gabapentin is a novel analgesic whose mechanism of action is not known. We investigated in a postoperative pain model whether adenosine triphosphate (ATP)-sensitive K+ (K(ATP)) channels, N-methyl-d-aspartic acid (NMDA) receptors, and Ca2+ channels are involved in the antiallodynic effect of intrathecal gabapentin. Mechanical allodynia was induced by a paw incision in isoflurane-anesthetized rats. Withdrawal thresholds to von Frey filament stimulation near the incision site were measured before and after incision and after intrathecal drug administration. The antiallodynic effect of gabapentin (100 mug) was not affected by intrathecal pretreatment with antagonists of K(ATP) channels, NMDA receptors or gamma-aminobutyric acid (GABA)(A) receptors. K(ATP) channel openers and GABA(A) receptor agonist, per se, had little effect on the postincision allodynic response. The Ca2+ channel blocker of N-type (omega-conotoxin GVIA, 0.1-3 microg), but not of P/Q-type (omega-agatoxin IVA), L-type (verapamil, diltiazem or nimodipine), or T-type (mibefradil), attenuated the incision-induced allodynia, as did gabapentin. Both the antiallodynic effects of gabapentin and omega-conotoxin GVIA were attenuated by Bay K 8644, an L-type Ca2+ channel activator. These results provide correlative evidence to support the contention that N-type Ca2+ channels, but not K(ATP) channels or NMDA or GABA(A) receptors, might be involved in the antiallodynic effect of intrathecal gabapentin.

  13. Role of KATP Channels in Beneficial Effects of Exercise in Ischemic Heart Failure.

    PubMed

    Kraljevic, Jasenka; Høydal, Morten Andre; Ljubkovic, Marko; Moreira, Jose Bianco Nascimento; Jørgensen, Kari; Ness, Henning Ofstad; Bækkerud, Fredrik Hjulstad; Dujic, Zeljko; Wisløff, Ulrik; Marinovic, Jasna

    2015-12-01

    Exercise training reduces pathological remodeling and improves cardiac function in ischemic heart failure; however, causal mechanisms underlying the cardiac benefits of exercise are poorly understood. Because opening of adenosine triphosphate (ATP)-sensitive K ⁺(KATP) channels protects the heart during myocardial stress, we hypothesized that such a mechanism is responsible for some of the cardiac benefits induced by exercise in postinfarction chronic heart failure (CHF). Left ventricular myocytes were isolated from three groups of rats: Sham, CHF Tr (4 wk after myocardial infarction, rats underwent 8 wk of aerobic interval training 5 d·wk⁻¹) and CHF Sed (rats sedentary for 12 wk after infarction). Cardiomyocyte survival after oxidative stress exposure (200 μM H2O2) and calcium handling (cells loaded with Fura-2 AM and electrically paced at 1 Hz) were assessed in the presence of KATP channel inhibitor glibenclamide. Expression of KATP subunits (SUR2A and Kir6.2) was evaluated using immunoblotting. Exercise improved cardiac function in CHF Tr animals. Cardiomyocytes from CHF Sed rats were more susceptible to oxidative stress-induced cell death than CHF Tr and Sham cardiomyocytes, with glibenclamide completely abolishing the protective effect of exercise. Glibenclamide did not affect cardiomyocyte survival in Sham or CHF Sed rats. In addition, exercise increased the systolic Ca²⁺ transient amplitude and improved diastolic Ca²⁺ removal in CHF Tr cardiomyocytes (compared with CHF Sed); both were significantly attenuated by glibenclamide. Exercise resulted in increased expression of KATP channel subunits in CHF Tr hearts, with more pronounced and significant effect on SUR2A. Our data suggest that KATP channel upregulation induced by chronic exercise likely mediates some of exercise-induced beneficial effects on cardiac function in postischemic heart failure.

  14. 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.

  15. Endogenous adenosine mediates coronary vasodilation during exercise after K(ATP)+ channel blockade.

    PubMed Central

    Duncker, D J; van Zon, N S; Pavek, T J; Herrlinger, S K; Bache, R J

    1995-01-01

    The mechanism of coronary vasodilation produced by exercise is not understood completely. Recently, we reported that blockade of vascular smooth muscle K(ATP)+ channels decreased coronary blood flow at rest, but did not attenuate the increments in coronary flow produced by exercise. Adenosine is not mandatory for maintaining basal coronary flow, or the increase in flow produced by exercise during normal arterial inflow, but does contribute to coronary vasodilation in hypoperfused myocardium. Therefore, we investigated whether adenosine opposed the hypoperfusion produced by K(ATP)+ channel blockade, thereby contributing to coronary vasodilation during exercise. 11 dogs were studied at rest and during exercise under control conditions, during intracoronary infusion of the K(ATP)+ channel blocker glibenclamide (50 micrograms/kg per min), and during intracoronary glibenclamide in the presence of adenosine receptor blockade. Glibenclamide decreased resting coronary blood flow from 45 +/- 5 to 35 +/- 4 ml/min (P < 0.05), but did not prevent exercise-induced increases of coronary flow. Glibenclamide caused an increase in myocardial oxygen extraction at the highest level of exercise with a decrease in coronary venous oxygen tension from 15.5 +/- 0.7 to 13.6 +/- 0.8 mmHg (P < 0.05). The addition of the adenosine receptor antagonist 8-phenyltheophylline (5 mg/kg intravenous) to K(ATP)+ channel blockade did not further decrease resting coronary blood flow but did attenuate the increase in coronary flow produced by exercise. This was accompanied by a further decrease of coronary venous oxygen tension to 10.1 +/- 0.7 mmHg (P < 0.05), indicating aggravation of the mismatch between oxygen demand and supply. These findings are compatible with the hypothesis that K+ATP channels modulate coronary vasomotor tone both under resting conditions and during exercise. However, when K(ATP)+ channels are blocked, adenosine released from the hypoperfused myocardium provides an alternate

  16. A role for ATP-sensitive potassium channels in male sexual behavior.

    PubMed

    McDevitt, Melissa A; Thorsness, Robert J; Levine, Jon E

    2009-02-01

    ATP-sensitive potassium (K(+)(ATP)) channels regulate cell excitability and are expressed in steroid-responsive brain regions involved in sexual behavior, such as the preoptic area (POA) and medial basal hypothalamus (MBH). We hypothesized that K(+)(ATP) channels serve as a mechanism by which testosterone can control the electrical activity of neurons and consequently elicit male sexual responsiveness. RT-PCR analysis indicated that castration induces, while testosterone inhibits, mRNA expression of the K(+)(ATP) channel subunit Kir6.2 in both the POA and MBH of adult male rats. Intracerebral infusion of the pharmacological K(+)(ATP) channel inhibitor tolbutamide increased the proportion of long-term castrates displaying sexual behavior and restored mount frequency, intromission frequency, and copulatory efficacy to values observed in testes-intact animals. Infusions of tolbutamide, but not vehicle, also decreased latencies to mount and intromit in castrated males. Unilateral tolbutamide infusion directly into the POA significantly reduced mount latency of castrates; however, it did not affect other copulatory measures, suggesting that blockade of K(+)(ATP) channels in additional brain regions may be necessary to recover the full range of sexual behavior. These data indicate that blockade of K(+)(ATP) channels is sufficient to elicit the male sexual response in the absence of testosterone. Our observations are consistent with the hypothesis that testosterone modulates male sexual behavior by regulating K(+)(ATP) channels in the brain. Decreased channel expression or channel blockade may increase the excitability of androgen-target neurons, rendering them more sensitive to the hormonal, chemical, and somatosensory inputs they receive, and potentially increase secretion of neurotransmitters that facilitate sexual behavior.

  17. Role of ATP-sensitive potassium channels in modulating nociception in rat model of bone cancer pain.

    PubMed

    Xia, Hui; Zhang, Dengwen; Yang, Shijie; Wang, Yu; Xu, Lin; Wu, Jinjing; Ren, Jing; Yao, Wenlong; Fan, Longchang; Zhang, Chuanhan; Tian, Yuke; Pan, Hui-Lin; Wang, Xueren

    2014-03-20

    Bone cancer pain is a major clinical problem and remains difficult to treat. ATP-sensitive potassium (KATP) channels may be involved in regulating nociceptive transmission at the spinal cord level. We determined the role of spinal KATP channels in the control of mechanical hypersensitivity in a rat model of bone cancer pain. The rat model of bone cancer pain was induced by implanting rat mammary gland carcinoma cells (Walker256) into the tibias. KATP modulators (pinacidil and glibenclamide) or the specific Kir6.2-siRNA were injected via an intrathecal catheter. The mechanical withdrawal threshold of rats was tested using von Frey filaments. The Kir6.2 mRNA and protein levels were measured by quantitative PCR and western blots, respectively. Intrathecal injection of pinacidil, a KATP channel opener, significantly increased the tactile withdrawal threshold of cancer cell-injected rats in a dose-dependent manner. In contrast, intrathecal delivery of glibenclamide, a KATP channel blocker, or the specific Kir6.2-siRNA significantly reduced the tactile withdrawal threshold of cancer cell-injected rats. The mRNA and protein levels of Kir6.2 in the spinal cord of cancer cell-injected rats were significantly lower than those in control rats. Our findings suggest that the KATP channel expression level in the spinal cord is reduced in bone cancer pain. Activation of KATP channels at the spinal level reduces pain hypersensitivity associated with bone cancer pain.

  18. Glucose elicits cephalic-phase insulin release in mice by activating KATP channels in taste cells.

    PubMed

    Glendinning, John I; Frim, Yonina G; Hochman, Ayelet; Lubitz, Gabrielle S; Basile, Anthony J; Sclafani, Anthony

    2017-04-01

    The taste of sugar elicits cephalic-phase insulin release (CPIR), which limits the rise in blood glucose associated with meals. Little is known, however, about the gustatory mechanisms that trigger CPIR. We asked whether oral stimulation with any of the following taste stimuli elicited CPIR in mice: glucose, sucrose, maltose, fructose, Polycose, saccharin, sucralose, AceK, SC45647, or a nonmetabolizable sugar analog. The only taste stimuli that elicited CPIR were glucose and the glucose-containing saccharides (sucrose, maltose, Polycose). When we mixed an α-glucosidase inhibitor (acarbose) with the latter three saccharides, the mice no longer exhibited CPIR. This revealed that the carbohydrates were hydrolyzed in the mouth, and that the liberated glucose triggered CPIR. We also found that increasing the intensity or duration of oral glucose stimulation caused a corresponding increase in CPIR magnitude. To identify the components of the glucose-specific taste-signaling pathway, we examined the necessity of Calhm1, P2X2+P2X3, SGLT1, and Sur1. Among these proteins, only Sur1 was necessary for CPIR. Sur1 was not necessary, however, for taste-mediated attraction to sugars. Given that Sur1 is a subunit of the ATP-sensitive K(+) channel (KATP) channel and that this channel functions as a part of a glucose-sensing pathway in pancreatic β-cells, we asked whether the KATP channel serves an analogous role in taste cells. We discovered that oral stimulation with drugs known to increase (glyburide) or decrease (diazoxide) KATP signaling produced corresponding changes in glucose-stimulated CPIR. We propose that the KATP channel is part of a novel signaling pathway in taste cells that mediates glucose-induced CPIR. Copyright © 2017 the American Physiological Society.

  19. Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels.

    PubMed Central

    Rainbow, Richard D; James, Marian; Hudman, Diane; Al Johi, Mohammed; Singh, Harprit; Watson, Peter J; Ashmole, Ian; Davies, Noel W; Lodwick, David; Norman, Robert I

    2004-01-01

    Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels. PMID:14672537

  20. Melatonin modulates permeability transition pore and 5-hydroxydecanoate induced KATP channel inhibition in isolated brain mitochondria.

    PubMed

    Waseem, Mohammad; Tabassum, Heena; Parvez, Suhel

    2016-11-01

    There is increasing recognition of the magnitude of mitochondria in neurodegenerative disorders. Mitochondria play a key role in apoptotic and necrotic cell death. Melatonin (Mel), an indoleamine produced in several organs including the pineal gland has been known for its neuroprotective actions. In our study, we have investigated whether the mitochondrial ATP sensitive potassium (mtKATP) channel blocker 5-hydroxydecanoate (5-HD) and calcium (Ca(2+)) affects permeability transition pore (PTP) alterations in isolated brain mitochondria treated with melatonin (Mel) and cyclosporin A (CsA). Mitochondrial swelling, mitochondrial membrane potential (Δψm), ROS measurement and mitochondrial respiration were evaluated in isolated brain mitochondria. In our results, mitochondrial swelling stimulated by exposing Ca(2+) ions and 5-HD associated by mPTP opening as depicted by modulation of CsA and Mel. In addition, Ca(2+) and 5-HD decreased Δψm, depleted intracellular ROS, and inhibition of mitochondrial respiration (state 3 and state 4) in isolated brain mitochondria. Addition of Mel and CsA has shown significant restoration in mitochondrial swelling, Δψm, intracellular ROS measurement and mitochondrial respiration in isolated brain mitochondria. Therefore, we speculate the modulatory effect of Mel and CsA in mitochondria treated with 5-HD and Ca(2+) hinders the mPTP-mediated mitochondrial dysfunction and cellular oxidative stress. We conclude that inhibition of mPT is one likely mechanism of CsA's and its neuroprotective actions. Development of neuroprotective agents including Mel targeting the mPTP therefore bears hope for future treatment of severe neurodegenerative diseases. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  1. α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

    PubMed Central

    Geng, Xuehui; Lou, Haiyan; Wang, Jian; Li, Lehong; Swanson, Alexandra L.; Sun, Ming; Beers-Stolz, Donna; Watkins, Simon; Perez, Ruth G.

    2011-01-01

    α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases. PMID:20858756

  2. Ventricular action potential adaptation to regular exercise: role of β-adrenergic and KATP channel function.

    PubMed

    Wang, Xinrui; Fitts, Robert H

    2017-08-01

    Regular exercise training is known to affect the action potential duration (APD) and improve heart function, but involvement of β-adrenergic receptor (β-AR) subtypes and/or the ATP-sensitive K(+) (KATP) channel is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to voluntary wheel-running or control groups; they were anesthetized after 6-8 wk of training, and myocytes were isolated. Exercise training significantly increased APD of apex and base myocytes at 1 Hz and decreased APD at 10 Hz. Ca(2+) transient durations reflected the changes in APD, while Ca(2+) transient amplitudes were unaffected by wheel running. The nonselective β-AR agonist isoproterenol shortened the myocyte APD, an effect reduced by wheel running. The isoproterenol-induced shortening of APD was largely reversed by the selective β1-AR blocker atenolol, but not the β2-AR blocker ICI 118,551, providing evidence that wheel running reduced the sensitivity of the β1-AR. At 10 Hz, the KATP channel inhibitor glibenclamide prolonged the myocyte APD more in exercise-trained than control rats, implicating a role for this channel in the exercise-induced APD shortening at 10 Hz. A novel finding of this work was the dual importance of altered β1-AR responsiveness and KATP channel function in the training-induced regulation of APD. Of physiological importance to the beating heart, the reduced response to adrenergic agonists would enhance cardiac contractility at resting rates, where sympathetic drive is low, by prolonging APD and Ca(2+) influx; during exercise, an increase in KATP channel activity would shorten APD and, thus, protect the heart against Ca(2+) overload or inadequate filling.NEW & NOTEWORTHY Our data demonstrated that regular exercise prolonged the action potential and Ca(2+) transient durations in myocytes isolated from apex and base regions at 1-Hz and shortened both at 10-Hz stimulation. Novel findings were that wheel running shifted the

  3. 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

  4. Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

    PubMed

    Martin, Gregory M; Rex, Emily A; Devaraneni, Prasanna; Denton, Jerod S; Boodhansingh, Kara E; DeLeon, Diva D; Stanley, Charles A; Shyng, Show-Ling

    2016-10-14

    ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the KATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Pharmacology of cardiac potassium channels.

    PubMed

    Li, Gui-Rong; Dong, Ming-Qing

    2010-01-01

    Cardiac K(+) channels are cardiomyocyte membrane proteins that regulate K(+) ion flow across the cell membrane on the electrochemical gradient and determine the resting membrane potential and the cardiac action potential morphology and duration. Several K(+) channels have been well studied in the human heart. They include the transient outward K(+) current I(to1), the ultra-rapidly activating delayed rectifier current I(Kur), the rapidly and slowly activating delayed rectifier currents I(Kr) and I(Ks), the inward rectifier K(+) current I(K1), and ligand-gated K(+) channels, including adenosine-5'-triphosphate (ATP)-sensitive K(+) current (I(KATP)) and acetylcholine-activated current (I(KACh)). Regional differences of K(+) channel expression contribute to the variable morphologies and durations of cardiac action potentials from sinus node and atrial to ventricular myocytes, and different ventricular layers from endocardium and midmyocardium to epicardium. They also show different responses to endogenous regulators and/or pharmacological agents. K(+) channels are well-known targets for developing novel anti-arrhythmic drugs that can effectively prevent/inhibit cardiac arrhythmias. Especially, atrial-specific K(+) channel currents (I(Kur) and I(KACh)) are the targets for developing atrial-selective anti-atrial fibrillation drugs, which has been greatly progressed in recent years. This chapter concentrates on recent advances in intracellular signaling regulation and pharmacology of cardiac K(+) channels under physiological and pathophysiological conditions.

  6. DPPX potassium channel antibody

    PubMed Central

    Tobin, William Oliver; Lennon, Vanda A.; Komorowski, Lars; Probst, Christian; Clardy, Stacey Lynn; Aksamit, Allen J.; Appendino, Juan Pablo; Lucchinetti, Claudia F.; Matsumoto, Joseph Y.; Pittock, Sean J.; Sandroni, Paola; Tippmann-Peikert, Maja; Wirrell, Elaine C.

    2014-01-01

    Objective: To describe the detection frequency and clinical associations of immunoglobulin G (IgG) targeting dipeptidyl-peptidase-like protein-6 (DPPX), a regulatory subunit of neuronal Kv4.2 potassium channels. Methods: Specimens from 20 patients evaluated on a service basis by tissue-based immunofluorescence yielded a synaptic immunostaining pattern consistent with DPPX-IgG (serum, 20; CSF, all 7 available). Transfected HEK293 cell-based assay confirmed DPPX specificity in all specimens. Sixty-nine patients with stiff-person syndrome and related disorders were also evaluated by DPPX-IgG cell-based assay. Results: Of 20 seropositive patients, 12 were men; median symptom onset age was 53 years (range, 13–75). Symptom onset was insidious in 15 and subacute in 5. Twelve patients reported prodromal weight loss. Neurologic disorders were multifocal. All had one or more brain or brainstem manifestations: amnesia (16), delirium (8), psychosis (4), depression (4), seizures (2), and brainstem disorders (15; eye movement disturbances [8], ataxia [7], dysphagia [6], dysarthria [4], respiratory failure [3]). Nine patients reported sleep disturbance. Manifestations of central hyperexcitability included myoclonus (8), exaggerated startle (6), diffuse rigidity (6), and hyperreflexia (6). Dysautonomia involved the gastrointestinal tract (9; diarrhea [6], gastroparesis, and constipation [3]), bladder (7), cardiac conduction system (3), and thermoregulation (1). Two patients had B-cell neoplasms: gastrointestinal lymphoma (1), and chronic lymphocytic leukemia (1). Substantial neurologic improvements followed immunotherapy in 7 of 11 patients with available treatment data. DPPX-IgG was not detected in any of the stiff-person syndrome patients. Conclusions: DPPX-IgG is a biomarker for an immunotherapy-responsive multifocal neurologic disorder of the central and autonomic nervous systems. PMID:25320100

  7. The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity.

    PubMed

    Lund, Trine M; Ploug, Kenneth B; Iversen, Anne; Jensen, Anders A; Jansen-Olesen, Inger

    2015-03-01

    Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β-hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β-hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release. Energy metabolism and neurotransmission are linked and involve ATP-sensitive potassium (KATP ) channels. However, it is still unclear how and to what degree available energy substrate affects this link. We investigated the effect of changing energy substrate from only glucose to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release.

  8. 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. Copyright © 2016 the American Physiological Society.

  9. Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel complex

    PubMed Central

    Kang, Minho; Hashimoto, Atsushi; Gade, Aravind

    2014-01-01

    Hydrogen sulfide (H2S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H2S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K+ (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H2S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H2S in inflammation. PMID:25552582

  10. 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

  11. ATP-sensitive potassium channels mediate contraction-induced attenuation of sympathetic vasoconstriction in rat skeletal muscle.

    PubMed

    Thomas, G D; Hansen, J; Victor, R G

    1997-06-01

    Sympathetic vasoconstriction is sensitive to inhibition by metabolic events in contracting rat and human skeletal muscle, but the underlying cellular mechanisms are unknown. In rats, this inhibition involves mainly alpha2-adrenergic vasoconstriction, which relies heavily on Ca2+ influx through voltage-dependent Ca2+ channels. We therefore hypothesized that contraction-induced inhibition of sympathetic vasoconstriction is mediated by ATP-sensitive potassium (KATP) channels, a hyperpolarizing vasodilator mechanism that could be activated by some metabolic product(s) of skeletal muscle contraction. We tested this hypothesis in anesthetized rats by measuring femoral artery blood flow responses to lumbar sympathetic nerve stimulation or intraarterial hindlimb infusion of the specific alpha2-adrenergic agonist UK 14,304 during KATP channel activation with diazoxide in resting hindlimb and during KATP channel block with glibenclamide in contracting hindlimb. The major new findings are twofold. First, like muscle contraction, pharmacologic activation of KATP channels with diazoxide in resting hindlimb dose dependently attenuated the vasoconstrictor responses to either sympathetic nerve stimulation or intraarterial UK 14,304. Second, the large contraction-induced attenuation in sympathetic vasoconstriction elicited by nerve stimulation or UK 14,304 was partially reversed when the physiologic activation of KATP channels produced by muscle contraction was prevented with glibenclamide. We conclude that contraction-induced activation of KATP channels is a major mechanism underlying metabolic inhibition of sympathetic vasoconstriction in exercising skeletal muscle.

  12. Ischemic preconditioning in the hippocampus of a knockout mouse lacking SUR1-based K(ATP) channels.

    PubMed

    Muñoz, Alvaro; Nakazaki, Mitsuhiro; Goodman, J Clay; Barrios, Roberto; Onetti, Carlos G; Bryan, Joseph; Aguilar-Bryan, Lydia

    2003-01-01

    ATP-sensitive K+ (K(ATP)) channels have been implicated in the mechanism of neuronal ischemic preconditioning. To evaluate the role of neuronal/beta-cell-type K(ATP) channels, SUR1 null (Sur1KO) mice lacking (K(IR)6.x/SUR1)(4) K(ATP) channels were subjected to a preconditioning protocol with the use of double carotid occlusion. Wild-type C57BL/6 and Sur1KO mice were subjected to a double carotid block for 40 minutes with or without a 20-minute preconditioning block. After a 10-day reperfusion period, damage was assessed histologically in the hippocampal CA1, CA2, and CA3 areas and in the dentate gyrus. The neuroprotective effects of intracerebroventricular injections of diazoxide, which selectively affects mitochondria versus opening SUR1-type K(ATP) channels, and 5-hydroxydecanoate, a selective blocker of mitoK(ATP) channels, were evaluated with the same protocol. Neurons in the CA1 region of both Sur1KO and wild-type animals subjected to a 20-minute ischemic insult were protected equally from neuronal damage produced by a subsequent 40-minute ischemic period. Pretreatment with diazoxide protected both Sur1KO and wild-type neurons, while 5-hydroxydecanoate augmented neurodegeneration in both strains of animals when administered before a 20-minute bout of ischemia. SUR1-based K(ATP) channels are not obligatory for neuronal preconditioning or augmentation of neurodegeneration by 5-hydroxydecanoate.

  13. Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes

    PubMed Central

    Arakel, Eric C.; Brandenburg, Sören; Uchida, Keita; Zhang, Haixia; Lin, Yu-Wen; Kohl, Tobias; Schrul, Bianca; Sulkin, Matthew S.; Efimov, Igor R.; Nichols, Colin G.; Lehnart, Stephan E.; Schwappach, Blanche

    2014-01-01

    ABSTRACT The copy number of membrane proteins at the cell surface is tightly regulated. Many ion channels and receptors present retrieval motifs to COPI vesicle coats and are retained in the early secretory pathway. In some cases, the interaction with COPI is prevented by binding to 14-3-3 proteins. However, the functional significance of this antagonism between COPI and 14-3-3 in terminally differentiated cells is unknown. Here, we show that ATP-sensitive K+ (KATP) channels, which are composed of Kir6.2 and SUR1 subunits, are stalled in the Golgi complex of ventricular, but not atrial, cardiomyocytes. Upon sustained β-adrenergic stimulation, which leads to activation of protein kinase A (PKA), SUR1-containing channels reach the plasma membrane of ventricular cells. We show that PKA-dependent phosphorylation of the C-terminus of Kir6.2 decreases binding to COPI and, thereby, silences the arginine-based retrieval signal. Thus, activation of the sympathetic nervous system releases this population of KATP channels from storage in the Golgi and, hence, might facilitate the adaptive response to metabolic challenges. PMID:24569881

  14. Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts

    PubMed Central

    Fedorov, Vadim V.; Glukhov, Alexey V.; Ambrosi, Christina M.; Kostecki, Geran; Chang, Roger; Janks, Deborah; Schuessler, Richard B.; Moazami, Nader; Nichols, Colin G.; Efimov, Igor R.

    2011-01-01

    INTRODUCTION This study compared the effects of ATP-regulated potassium channel (KATP) openers, diazoxide and pinacidil, on diseased and normal human atria and ventricles. METHODS We optically mapped the endocardium of coronary-perfused right (n=11) or left (n=2) posterior atrial-ventricular free wall preparations from human hearts with congestive heart failure (CHF, n=8) and non-failing human hearts without (NF, n=3) or with (INF, n=2) infarction. We also analyzed the mRNA expression of the KATP targets Kir6.1, Kir6.2, SUR1, and SUR2 in the left atria and ventricles of NF (n=8) and CHF (n=4) hearts. RESULTS In both CHF and INF hearts, diazoxide significantly decreased action potential durations (APDs) in atria (by −21±3% and −27±13%, p<0.01) and ventricles (by −28±7% and −28±4%, p<0.01). Diazoxide did not change APD (0±5%) in NF atria. Pinacidil significantly decreased APDs in both atria (−46 to - 80%, p<0.01) and ventricles (−65 to −93%, p<0.01) in all hearts studied. The effect of pinacidil on APD was significantly higher than that of diazoxide in both atria and ventricles of all groups (p<0.05). During pinacidil perfusion, burst pacing induced flutter/fibrillation in all atrial and ventricular preparations with dominant frequencies of 14.4±6.1 Hz and 17.5 ±5.1 Hz, respectively. Glibenclamide (10 μM) terminated these arrhythmias and restored APDs to control values. Relative mRNA expression levels of KATP targets were correlated to functional observations. CONCLUSION Remodeling in response to CHF and/or previous infarct potentiated diazoxide-induced APD shortening. The activation of atrial and ventricular KATP channels enhances arrhythmogenicity, suggesting that such activation may contribute to reentrant arrhythmias in ischemic hearts. PMID:21586291

  15. CD200 Inhibits Inflammatory Response by Promoting KATP Channel Opening in Microglia Cells in Parkinson’s Disease

    PubMed Central

    Ren, Yi; Ye, Min; Chen, Shengdi; Ding, Jianqing

    2016-01-01

    Background As the second most common neurodegenerative disorder after Alzheimer’s disease (AD), Parkinson’s disease (PD) principally impacts the motor system in approximately 7 million patients worldwide. The present study aimed to explore the effects of cluster of differentiation (CD200) on adenosine triphosphate-sensitive potassium (KATP) channels and inflammatory response in PD mice. Material/Methods We created an in vivo PD model by intraperitoneal injection of 30 mg/kg/day 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP. HCL) for 5 consecutive days, and we created an in vitro PD model by injection of 100 μM 1-methyl-4-phenylpyridinium ion (MPP+) in primary microglia cells. Expression level of CD200/CD200R, inwardly rectifying potassium (Kir6.1/6.2), and sulfonylurea receptor (Sur1/2) were detected by Western blot (WB). Immunohistochemistry (IHC) was utilized to assess CD11b (microglia marker) and tyrosine hydroxylase (TH, a marker reveals dopamine level in neurons) expression levels. An in vitro PD model was applied to detect the influence of CD200 on ATP and inflammatory factors released from microglia. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β mRNA levels were explored by realtime quantitative polymerase chain reaction (RT-QPCR), and their protein levels were identified by enzyme-linked immunosorbent assay (ELISA). Results WB exhibited time-dependent down-regulation of CD200/CD200R in cerebra of PD mice compared to control mice, with Kir 6.1 and SUR 2 expressed mainly in microglia. IHC showed that CD11b reached a peak at the 1st day after MPTP treatment, followed by time-dependent reduction, and TH decreased noticeably after MPTP induction. RT-QPCR demonstrated that compared with controls, IFN-γ, TNF-α, and IL-1β mRNA levels were significantly elevated at MPTP-1d, was reduced at MPTP-3d, and then returned to baseline at MPTP-7d. IHC showed that MPP+ significantly elevated microglia release of

  16. 1,4-Benzothiazine ATP-sensitive potassium channel openers: modifications at the C-2 and C-6 positions.

    PubMed

    Martelli, Alma; Manfroni, Giuseppe; Sabbatini, Paola; Barreca, Maria Letizia; Testai, Lara; Novelli, Michela; Sabatini, Stefano; Massari, Serena; Tabarrini, Oriana; Masiello, Pellegrino; Calderone, Vincenzo; Cecchetti, Violetta

    2013-06-13

    ATP-sensitive potassium (KATP) channels play a prominent role in controlling cardiovascular function. In this paper, a novel series of 4-(1-oxo-2-cyclopentenyl)-1,4-benzothiazine derivatives modified at the C-2, and C-6 positions were synthesized as openers of vascular KATP channels. Most of the tested compounds evoked vasorelaxing effects on rat aortic rings and membrane hyperpolarization in human vascular smooth muscle cells, with potency similar or superior to that of the reference levcromakalim (LCRK). The selective KATP blocker glibenclamide antagonized the above vascular effects, confirming that KATP channels are closely involved in the mechanism of action. The experimental results confirmed the 1,4-benzothiazine nucleus as an optimal scaffold for activators of vascular KATP channels; moreover, the high level of potency exhibited by the 6-acetyl substituted benzothiazine 8, along with the lack of any significant interference with insulin secretion from pancreatic β-cells, paves the way to further develop a new series of potent activators of vascular KATP channels.

  17. Genetic Control of Potassium Channels.

    PubMed

    Amin, Ahmad S; Wilde, Arthur A M

    2016-06-01

    Approximately 80 genes in the human genome code for pore-forming subunits of potassium (K(+)) channels. Rare variants (mutations) in K(+) channel-encoding genes may cause heritable arrhythmia syndromes. Not all rare variants in K(+) channel-encoding genes are necessarily disease-causing mutations. Common variants in K(+) channel-encoding genes are increasingly recognized as modifiers of phenotype in heritable arrhythmia syndromes and in the general population. Although difficult, distinguishing pathogenic variants from benign variants is of utmost importance to avoid false designations of genetic variants as disease-causing mutations.

  18. Possible involvement of ATP-sensitive potassium channels in the antidepressant-like effects of gabapentin in mouse forced swimming test.

    PubMed

    Ostadhadi, Sattar; Akbarian, Reyhaneh; Norouzi-Javidan, Abbas; Nikoui, Vahid; Zolfaghari, Samira; Chamanara, Mohsen; Dehpour, Ahmad-Reza

    2017-07-01

    Gabapentin as an anticonvulsant drug also has beneficial effects in treatment of depression. Previously, we showed that acute administration of gabapentin produced an antidepressant-like effect in the mouse forced swimming test (FST) by a mechanism that involves the inhibition of nitric oxide (NO). Considering the involvement of NO in adenosine triphosphate (ATP)-sensitive potassium channels (KATP), in the present study we investigated the involvement of KATP channels in antidepressant-like effect of gabapentin. Gabapentin at different doses (5-10 mg/kg) and fluoxetine (20 mg/kg) were administrated by intraperitoneal route, 60 and 30 min, respectively, before the test. To clarify the probable involvement of KATP channels, mice were pretreated with KATP channel inhibitor or opener. Gabapentin at dose 10 mg/kg significantly decreased the immobility behavior of mice similar to fluoxetine (20 mg/kg). Co-administration of subeffective dose (1 mg/kg) of glibenclamide (inhibitor of KATP channels) with gabapentin (3 mg/kg) showed a synergistic antidepressant-like effect. Also, subeffective dose of cromakalim (opener of KATP channels, 0.1 mg/kg) inhibited the antidepressant-like effect of gabapentin (10 mg/kg). None of the treatments had any impact on the locomotor movement. Our study, for the first time, revealed that antidepressant-like effect of gabapentin in mice is mediated by blocking the KATP channels.

  19. Potassium channels and their evolving gates.

    PubMed

    Jan, L Y; Jan, Y N

    1994-09-08

    Potassium channels allow potassium ions to flow across the membrane and play a key role in maintaining membrane potential. Recent research has begun to reveal how these channels transport potassium in preference to other ions, how their activity is controlled, and how they are related to other channels.

  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. Intramolecular interaction of SUR2 subtypes for intracellular ADP-Induced differential control of K(ATP) channels.

    PubMed

    Matsushita, Kenji; Kinoshita, Kengo; Matsuoka, Tetsuro; Fujita, Akikazu; Fujikado, Takashi; Tano, Yasuo; Nakamura, Haruki; Kurachi, Yoshihisa

    2002-03-22

    ATP-sensitive K+ (K(ATP)) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K(ATP) channels in SUR-subtypes. By examining ADP-effect on K(ATP) channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K(ATP) channels.

  2. Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes

    PubMed Central

    Zhang, Quan; Ramracheya, Reshma; Lahmann, Carolina; Tarasov, Andrei; Bengtsson, Martin; Braha, Orit; Braun, Matthias; Brereton, Melissa; Collins, Stephan; Galvanovskis, Juris; Gonzalez, Alejandro; Groschner, Lukas N.; Rorsman, Nils J.G.; Salehi, Albert; Travers, Mary E.; Walker, Jonathan N.; Gloyn, Anna L.; Gribble, Fiona; Johnson, Paul R.V.; Reimann, Frank; Ashcroft, Frances M.; Rorsman, Patrik

    2013-01-01

    Summary Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes. PMID:24315372

  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. 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. © 2013 The British Pharmacological Society.

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

    PubMed Central

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (KATP) 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 KATP 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. PMID:24102106

  6. ATP-sensitive inwardly rectifying potassium channel regulation of viral infections in honey bees.

    PubMed

    O'Neal, Scott T; Swale, Daniel R; Anderson, Troy D

    2017-08-17

    Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. In the work described here, we have demonstrated the use of flock house virus (FHV) as a model system for virus infection in bees and revealed an important role for the regulation of the bee antiviral immune response by ATP-sensitive inwardly rectifying potassium (KATP) channels. We have shown that treatment with the KATP channel agonist pinacidil increases survival of bees while decreasing viral replication following infection with FHV, whereas treatment with the KATP channel antagonist tolbutamide decreases survival and increases viral replication. Our results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees.

  7. 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.

  8. 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

  9. Effects of acidosis and NO on nicorandil-activated KATP channels in guinea-pig ventricular myocytes

    PubMed Central

    Moncada, Gustavo A; Kishi, Yukio; Numano, Fujio; Hiraoka, Masayasu; Sawanobori, Tohru

    2000-01-01

    Nicorandil is a hybrid compound of K+ channel opener and nitrate. We investigated a possible interaction of acidosis and nitric oxide (NO)-donors on the nicorandil-activated ATP-sensitive K+ channel (KATP) in guinea-pig ventricular myocytes using the patch-clamp technique.In whole-cell recordings, external application of 300 μM nicorandil activated KATP in the presence of 2 mM intracellular ATP concentration ([ATP]i) at external pH (pHo) 7.4, but the activated current was decreased by reducing pHo to 6.5–6.0.Single-channel recordings of inside-out patches revealed decreased open-state probability (Po) of KATP activated by nicorandil with reducing internal pH (pHi) from 7.2 to 6.0, whilst the channel activity increased at low pHi in the absence of nicorandil.Application of NO donors, 1 mM-sodium nitroprusside (SNP) or -NOR-3 to the membrane cytoplasmic side at pHi 7.2 increased the channel activity but decreased it at pHi 6.5–6.0. Neither removal of the drugs nor application of NO-scavengers reversed depression of channel activity induced by NO-donors.We conclude that an increase in pHo and pHi depresses rather than stimulates the nicorandil-activated KATP. Since NO-donors at low pHi exhibited a similar trend, involvement of H+ and NO interaction can be considered as a mechanism of decreased KATP activated by nicorandil. PMID:11082116

  10. The roles of KCa, KATP, and KV channels in regulating cutaneous vasodilation and sweating during exercise in the heat.

    PubMed

    Louie, Jeffrey C; Fujii, Naoto; Meade, Robert D; McNeely, Brendan D; Kenny, Glen P

    2017-05-01

    We recently showed the varying roles of Ca(2+)-activated (KCa), ATP-sensitive (KATP), and voltage-gated (KV) K(+) channels in regulating cholinergic cutaneous vasodilation and sweating in normothermic conditions. However, it is unclear whether the respective contributions of these K(+) channels remain intact during dynamic exercise in the heat. Eleven young (23 ± 4 yr) men completed a 30-min exercise bout at a fixed rate of metabolic heat production (400 W) followed by a 40-min recovery period in the heat (35°C, 20% relative humidity). Cutaneous vascular conductance (CVC) and local sweat rate were assessed at four forearm skin sites perfused via intradermal microdialysis with: 1) lactated Ringer solution (control); 2) 50 mM tetraethylammonium (nonspecific KCa channel blocker); 3) 5 mM glybenclamide (selective KATP channel blocker); or 4) 10 mM 4-aminopyridine (nonspecific KV channel blocker). Responses were compared at baseline and at 10-min intervals during and following exercise. KCa channel inhibition resulted in greater CVC versus control at end exercise (P = 0.04) and 10 and 20 min into recovery (both P < 0.01). KATP channel blockade attenuated CVC compared with control during baseline (P = 0.04), exercise (all P ≤ 0.04), and 10 min into recovery (P = 0.02). No differences in CVC were observed with KV channel inhibition during baseline (P = 0.15), exercise (all P ≥ 0.06), or recovery (all P ≥ 0.14). With the exception of KV channel inhibition augmenting sweating during baseline (P = 0.04), responses were similar to control with all K(+) channel blockers during each time period (all P ≥ 0.07). We demonstrated that KCa and KATP channels contribute to the regulation of cutaneous vasodilation during rest and/or exercise and recovery in the heat. Copyright © 2017 the American Physiological Society.

  11. Overexcited MaxiK and KATP channels underlie obstructive jaundice-induced vasoconstrictor hyporeactivity of arterial smooth muscle

    PubMed Central

    Yuan, Ya-wei; Wang, Long; Lu, Zhan-ying; Long, Yue; Jiao, Ying-fu; Xia, Qiang; Wen, Da-xiang; Yu, Wei-feng

    2016-01-01

    Substantial evidence has shown that obstructive jaundice can induce vascular hyporesponsiveness. The present study was designed to investigate mechanisms of MaxiK channel and KATP underlying cholestasis-induced vascular dysfunction. The isolated thoracic aorta was used to explore norepinephrine (NE)-induced contraction. The function of MaxiK and KATP channels were investigated using whole-cell patch clamp recording. Compared with Sham group, NE-induced vascular contraction was blunted after bile duct ligation (BDL), which could not be ameliorated significantly after endothelial denudation. Charybdotoxin and glibenclamide induced a more pronounced recovery from vascular hyporesponsiveness to NE in BDL group compared with Sham group. BDL significantly promoted the charybdotoxin sensitive MaxiK current and KATP current in isolated aortic smooth muscle cells. In addition, the expression of auxiliary subunits (MaxiK-β1 and SUR2B) rather pore-forming subunits (MaxiK-α and Kir6.1) was significantly up-regulated after BDL. These findings suggest that MaxiK and KATP channels play an important role in regulating vascular hyporesponsiveness in BDL rats. PMID:28000721

  12. Role of ATP-dependent potassium channels in pulmonary vascular tone of fetal lambs with congenital diaphragmatic hernia.

    PubMed

    de Buys Roessingh, Anthony S; de Lagausie, Pascal; Barbet, Jacques-Patrick; Mercier, Jean-Christophe; Aigrain, Yves; Dinh-Xuan, Anh Tuan

    2006-11-01

    High mortality in newborn babies with congenital diaphragmatic hernia (CDH) is principally due to persistent pulmonary hypertension. ATP-dependent potassium (K(ATP)) channels might modulate pulmonary vascular tone. We have assessed the effects of Pinacidil, a K(ATP) channel opener, and glibenclamide (GLI), a K(ATP) channel blocker, in near full-term lambs with and without CDH. In vivo, pulmonary hemodynamics were assessed by means of pressure and blood flow catheters. In vitro, we used isolated pulmonary vessels and immunohistochemistry to detect the presence of K(ATP) channels in pulmonary tissue. In vivo, pinacidil (2 mg) significantly reduced pulmonary vascular resistance (PVR) in both controls and CDH animals. GLI (30 mg) significantly increased pulmonary arterial pressure (PAP) and PVR in control animals only. In vitro, pinacidil (10 microM) relaxed, precontracted arteries from lambs with and without CDH. GLI (10(-5) microM) did not raise the basal tone of vessels. We conclude that activation of K(ATP) channels could be of interest to reduce pulmonary vascular tone in fetal lambs with CDH, a condition often associated with persistent pulmonary hypertension of the newborn.

  13. Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel

    PubMed Central

    Sun, Haiyan; Wei, Ying; Deng, Huayun; Xiong, Qiaojie; Li, Min; Lahiri, Joydeep; Fang, Ye

    2014-01-01

    Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel. PMID:24816792

  14. Structure and dynamics of the pore of inwardly rectifying K(ATP) channels.

    PubMed

    Loussouarn, G; Makhina, E N; Rose, T; Nichols, C G

    2000-01-14

    Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.

  15. Hydrogen sulfide attenuates gastric mucosal injury induced by restraint water-immersion stress via activation of KATP channel and NF-κB dependent pathway

    PubMed Central

    Sun, Hong-Zhao; Zheng, Shan; Lu, Kai; Hou, Feng-Tian; Bi, Jie-Xue; Liu, Xue-Lian; Wang, Shan-Shan

    2017-01-01

    AIM To explore the effect of hydrogen sulfide (H2S) on restraint water-immersion stress (RWIS)-induced gastric lesions in rats and the influence of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway on such an effect. METHODS Male Wistar rats were randomly divided into a control group, a physiological saline (PS) group, a sodium hydrosulfide (NaHS) group, a glibenclamide (Gl) group, Gl plus NaHS group, a pyrrolidine dithiocarbamate (PDTC) group, and a PDTC plus NaHS group. Gastric mucosal injury was induced by RWIS for 3 h in rats, and gastric mucosal damage was analyzed after that. The PS, NaHS (100 μmol/kg body weight), Gl (100 μmol/kg body weight), Gl (100 μmol/kg or 150 μmol/kg body weight) plus NaHS (100 μmol/kg body weight), PDTC (100 μmol/kg body weight), and PDTC (100 μmol/kg body weight) plus NaHS (100 μmol/kg body weight) were respectively injected intravenously before RWIS. RESULTS RWIS induced serious gastric lesions in the rats in the PS pretreatment group. The pretreatment of NaHS (a H2S donor) significantly reduced the damage induced by RWIS. The gastric protective effect of the NaHS during RWIS was attenuated by PDTC, an NF-κB inhibitor, and also by glibenclamide, an ATP-sensitive potassium channel blocker, in a dose-dependent manner. CONCLUSION These results suggest that exogenous H2S plays a protective role against RWIS injury in rats, possibly through modulation of KATP channel opening and the NF-κB dependent pathway. PMID:28104983

  16. Subclinical Doses of ATP-Sensitive Potassium Channel Modulators Prevent Alterations in Memory and Synaptic Plasticity Induced by Amyloid-β.

    PubMed

    Salgado-Puga, Karla; Rodríguez-Colorado, Javier; Prado-Alcalá, Roberto A; Peña-Ortega, Fernando

    2017-02-10

    In addition to coupling cell metabolism and excitability, ATP-sensitive potassium channels (KATP) are involved in neural function and plasticity. Moreover, alterations in KATP activity and expression have been observed in Alzheimer's disease (AD) and during amyloid-β (Aβ)-induced pathology. Thus, we tested whether KATP modulators can influence Aβ-induced deleterious effects on memory, hippocampal network function, and plasticity. We found that treating animals with subclinical doses (those that did not change glycemia) of a KATP blocker (Tolbutamide) or a KATP opener (Diazoxide) differentially restrained Aβ-induced memory deficit, hippocampal network activity inhibition, and long-term synaptic plasticity unbalance (i.e., inhibition of LTP and promotion of LTD). We found that the protective effect of Tolbutamide against Aβ-induced memory deficit was strong and correlated with the reestablishment of synaptic plasticity balance, whereas Diazoxide treatment produced a mild protection against Aβ-induced memory deficit, which was not related to a complete reestablishment of synaptic plasticity balance. Interestingly, treatment with both KATP modulators renders the hippocampus resistant to Aβ-induced inhibition of hippocampal network activity. These findings indicate that KATP are involved in Aβ-induced pathology and they heighten the potential role of KATP modulation as a plausible therapeutic strategy against AD.

  17. 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.

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

    PubMed

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

    2016-09-28

    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.

  19. 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

  20. A KATP-channel opener as a potential treatment modality for erectile dysfunction.

    PubMed

    Moon, D G; Byun, H S; Kim, J J

    1999-05-01

    To assess the effects of pinacidil (a KATP-channel opener) for the treatment of penile erectile dysfunction and to examine the role of the K+-channel in cavernosal smooth muscle contractility. Using a feline model, the magnitude of penile erection caused by pinacidil was compared with that caused by erectogenic drugs, e.g. acetylcholine, prostaglandin E1 (PGE1) and L-arginine. The effects of K+-channel blockers (4-aminopyridine, glibenclamide and tetraethylammonium) and pinacidil on penile erections induced by the drugs were investigated. The intra-arterial injection of pinacidil caused a dose-dependent increase in intracavernosal pressure (ICP) and the increase in ICP induced by pinacidil with acetylcholine, PGE1 or L-arginine was more pronounced than with the compounds alone. Furthermore, pinacidil (1 mmol/L) effectively reversed the inhibitory effects of the K+-channel blockers on the cavernosal relaxation induced by acetylcholine, PGE1 or L-arginine (P<0.01). Notably, pinacidil induced cavernosal relaxation after injecting the drugs even in cases refractory to higher concentrations (0.1 mol/L) of the drugs (n=11, P<0.01). These results suggest that pinacidil is effective in relaxing feline erectile tissue in vivo, probably via increased K+ permeability and subsequent hyperpolarization. Further comparative studies with erectogenic compounds on human erectile tissue and clinical testing are required to determine whether K+-channel openers can be used in the diagnosis and treatment of erectile dysfunction. However, pinacidil seems promising as an intracavernosal agent combined with PGE1 to produce synergistic effects.

  1. Role of ATP-sensitive potassium channels in the piracetam induced blockade of opioid effects.

    PubMed

    Rehni, Ashish K; Singh, Nirmal; Jindal, Seema

    2007-12-01

    The present study has been designed to investigate the effect of piracetam on morphine/ buprenorphine-induced antinociception in rats and effect of piracetam on morphine or minoxidil induced relaxation in KCl-precontracted isolated rat aortic ring preparation. Nociceptive threshold was measured by the tail flick test in rats. The cumulative dose responses of morphine or minoxidil were recorded in KCl-precontracted isolated rat aortic ring preparation. Piracetam attenuated buprenorphine-induced antinociception in rats. Piracetam significantly reduced the morphine and minoxidil induced relaxation in KCl precontracted isolated rat aortic ring preparation suggesting that piracetam interferes with opioid receptor and ATP-sensitive potassium channel (KATP) opener mediated responses in vitro. Thus, it may be suggested that piracetam attenuates opioid effects by an opioid receptor-KATP channel linked mechanism.

  2. Adenosine triphosphate-sensitive potassium channel blockers attenuate the antiallodynic effect of R-PIA in neuropathic rats.

    PubMed

    Song, Jun-Gol; Hahm, Kyung Don; Kim, Young Ki; Leem, Jeong Gil; Lee, Chung; Jeong, Sung Moon; Park, Pyung Hwan; Shin, Jin Woo

    2011-06-01

    Nerve injury can generate neuropathic pain. The accompanying mechanical allodynia may be reduced by the intrathecal administration of adenosine. The neuroprotective effects of adenosine are mediated by the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel. We assessed the relationship between the adenosine A1 receptor agonist, N⁶-(R)-phenylisopropyl adenosine (R-PIA), and K(ATP) channels to determine whether the antiallodynic effects of R-PIA are also mediated through K(ATP) channels in a rat nerve ligation injury model of neuropathic pain. Mechanical allodynia was induced by tight ligation of the left lumbar fifth and sixth spinal nerves. Mechanical allodynia in the left hindpaw was evaluated using von Frey filaments to measure withdrawal thresholds. R-PIA (0.5, 1, or 2 μg) was administered intrathecally to induce antiallodynia. We assessed whether pretreatment with the K(ATP) channel blockers glibenclamide or 5-hydroxydecanoate reversed the antiallodynic effect of R-PIA. Also, we evaluated whether diazoxide, a K(ATP) channel opener, had an antiallodynic effect and promoted the antiallodynic effect of R-PIA. Lastly, we investigated whether the voltage-activated K channel blocker 4-aminopyridine attenuated the effect of R-PIA. Intrathecal R-PIA produced maximal antiallodynia at 2 μg (P < 0.05). Intrathecal pretreatment with glibenclamide and intraperitoneal pretreatment 5-hydroxydecanoate significantly reduced the antiallodynic effect of R-PIA. Diazoxide produced an antiallodynic effect and also enhanced the antiallodynic action of R-PIA. 4-Aminopyridine had no effect on the antiallodynic action of R-PIA. The antiallodynic effects of adenosine A1 receptor stimulation may be related to K(ATP) channel activity in a rat model of nerve ligation injury.

  3. Calcium Activation of Mougeotia Potassium Channels 1

    PubMed Central

    Lew, Roger R.; Serlin, Bruce S.; Schauf, Charles L.; Stockton, Marsha E.

    1990-01-01

    Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca2+] as low as 20 micromolar. However, external [Ca2+] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation. PMID:16667356

  4. The scorpion toxin and the potassium channel.

    PubMed

    Swartz, Kenton J

    2013-05-21

    The structure of a complex containing a toxin bound to a potassium ion channel has been solved for the first time, revealing how scorpions have designed toxins that can recognize and target the filter that controls the movement of potassium ions through these channels.

  5. Imidazoline/guanidinium binding sites and their relation to inhibition of K(ATP) channels in pancreatic B-cells.

    PubMed

    Rustenbeck, I; Herrmann, C; Ratzka, P; Hasselblatt, A

    1997-09-01

    To elucidate the beta-cytotropic effect of imidazoline compounds their inhibitory effect on ATP-dependent K+ channels (K(ATP) channels) in pancreatic B-cells was compared with their binding to membranes from insulin-secreting HIT T15 cells. K(ATP) channels in inside-out patches from B-cells were closed with the following rank order of efficacy at 10 microM: guanabenz > phentolamine = alinidine > clonidine > idazoxan > rilmenidine = amiloride. The last four compounds achieved an incomplete inhibition only. In contrast to sulfonylureas, the inhibitory action of imidazolines was not enhanced by ADP. With intact cells the site which mediates inhibition is less easily accessible for protonated compounds, suggesting a location at the inner face of the plasma membrane. Competition binding experiments were performed by masking alpha-adrenoceptors and using [3H]clonidine as ligand. Homologous displacement of [3H]clonidine revealed two distinct binding sites in HIT cell membranes characterized by dissociation constants of 38 nM and 4,911 nM and maximal binding capacities of 118 fmol/mg protein and 18 pmol/mg protein. Generally, ligands for I2 imidazoline receptors were more potent than ligands for I1 imidazoline receptors to displace [3H]clonidine from the high affinity site, which does not fit into the current classification of imidazoline receptors. Binding to the second site had affinities in the micromolar range, similar to the concentrations necessary to inhibit K(ATP) channels in B-cells. However, alinidine and phentolamine inhibited K(ATP) channels already at concentrations at which they displaced [3H]clonidine only from the high affinity site, but not yet from the low affinity site. Since the proportion of the low and high affinity site varied in dependence of the competitor, the imidazoline binding sites in HIT cells may not be independent, but may rather represent two interacting or interconvertible sites both of which may be involved in K(ATP) channel closure.

  6. Reactive Oxygen Species and Mitochondrial KATP Channels Mediate Helium-Induced Preconditioning Against Myocardial Infarction In Vivo

    PubMed Central

    Pagel, Paul S.; Krolikowski, John G.; Pratt, Phillip F.; Shim, Yon Hee; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

    2008-01-01

    Objectives Helium produces preconditioning by activating prosurvival kinases, but the roles of reactive oxygen species (ROS) or mitochondrial KATP channels in this process are unknown. We tested the hypothesis that ROS and mitochondrial KATP channels mediate helium-induced preconditioning in vivo. Design Randomized, prospective study. Setting University research laboratory. Participants Male New Zealand white rabbits. Interventions Rabbits (n=64) were instrumented for measurement of systemic hemodynamics and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion. In separate experimental groups, rabbits (n=7 or 8 per group) were randomly assigned to receive 0.9% saline (control) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture before LAD occlusion with or without the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the mitochondrial KATP antagonist 5-hydroxydecanoate (5-HD; 5 mg/kg). Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni's modification of Student's t test. Measurements and Main Results Myocardial infarct size was determined using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium significantly (P<0.05) reduced infarct size (23±4% of the area at risk; mean±SD) compared with control (46±3%). NAC, 2-MPG, and 5-HD did not affect irreversible ischemic injury when administered alone (49±5, 45±6, and 45±3%), but these drugs blocked reductions in infarct size produced by helium (45±4, 45±2, and 44±3%). Conclusions The results suggest that ROS and mitochondrial KATP channels mediate helium-induced preconditioning in vivo. PMID:18662630

  7. Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets.

    PubMed

    Li, Changhong; Ackermann, Amanda M; Boodhansingh, Kara E; Bhatti, Tricia R; Liu, Chengyang; Schug, Jonathan; Doliba, Nicolai; Han, Bing; Cosgrove, Karen E; Banerjee, Indraneel; Matschinsky, Franz M; Nissim, Itzhak; Kaestner, Klaus H; Naji, Ali; Adzick, N Scott; Dunne, Mark J; Stanley, Charles A; De León, Diva D

    2017-07-01

    Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca(2+)] i ), insulin secretion, oxygen consumption, and [U-(13)C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca(2+)] i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-(13)C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in (13)C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked (13)C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition. © 2017 by the American Diabetes Association.

  8. Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism*

    PubMed Central

    Bushman, Jeremy D.; Gay, Joel W.; Tewson, Paul; Stanley, Charles A.; Shyng, Show-Ling

    2010-01-01

    The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (KATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in KATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for KATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues. PMID:20032456

  9. Minoxidil prevents 3,4-methylenedioxymethamphetamine-induced serotonin depletions: role of mitochondrial ATP-sensitive potassium channels, Akt and ERK.

    PubMed

    Goñi-Allo, Beatriz; Puerta, Elena; Ramos, María; Lasheras, Berta; Jordán, Joaquín; Aguirre, Norberto

    2008-02-01

    Preconditioning has emerged as a valid strategy against different neurotoxic insults. Although the mechanisms underlying preconditioning are not fully understood, the activation of ATP-sensitive potassium (KATP) channels has been proposed to play a pivotal role in neuronal preconditioning. In the present work we examine whether minoxidil a KATP channel activator protects against the long-term toxicity caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in rats. Our data show that intrastriatal administration of minoxidil prevents MDMA-induced long-term indole depletions in the rat striatum. This effect was not related to an effect on core temperature, as pre-treatment with minoxidil did not significantly alter MDMA-induced hyperthermia. Taking into account that minoxidil opens both sarcolemmal and mitochondrial KATP channels, we examined the role of each type of channels in the protective effects of minoxidil using specific inhibitors. The administration of HMR-1098, a blocker of the sarcolemmal KATP channels, along with minoxidil did not affect the protection afforded by the latter. On the contrary the selective mitochondrial KATP channel blocker 5-hydroxydecanoic acid completely reversed the protection afforded by minoxidil, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Furthermore our data show the participation of Akt and extracellular signal-regulated kinases in minoxidil-afforded protection. Intrastriatal administration of wortmannin or PD98059 (inhibitors of phosphatidylinositol-3-kinase and mitogen-activated protein kinase/extracellular regulated protein kinase, respectively), along with minoxidil abolished the protective effect of minoxidil against the serotonergic toxicity caused by MDMA. These results demonstrate that minoxidil by opening mitochondrial KATP channels completely prevents MDMA toxicity and that Akt and MAP kinases are involved in minoxidil-afforded neuroprotection.

  10. Mepivacaine attenuates vasodilation induced by ATP-sensitive potassium channels in rat aorta.

    PubMed

    Baik, Jiseok; Ok, Seong-Ho; Kim, Eun-Jin; Kang, Dawon; Hong, Jeong-Min; Shin, Il-Woo; Lee, Heon Keun; Chung, Young-Kyun; Cho, Youngil; Lee, Soo Hee; Kang, Sebin; Sohn, Ju-Tae

    2016-07-05

    The goal of this in vitro study was to investigate the effect of mepivacaine on vasodilation induced by the ATP-sensitive potassium (KATP) channel opener levcromakalim in isolated endothelium-denuded rat aortas. The effects of mepivacaine and the KATP channel inhibitor glibenclamide, alone or in combination, on levcromakalim-induced vasodilation were assessed in the isolated aortas. The effects of mepivacaine or combined treatment with a protein kinase C (PKC) inhibitor, GF109203X, and mepivacaine on this vasodilation were also investigated. Levcromakalim concentration-response curves were generated for isolated aortas precontracted with phenylephrine or a PKC activator, phorbol 12,13-dibutyrate (PDBu). Further, the effects of mepivacaine and glibenclamide on levcromakalim-induced hyperpolarization were assessed in rat aortic vascular smooth muscle cells. Mepivacaine attenuated levcromakalim-induced vasodilation, whereas it had no effect on this vasodilation in isolated aortas pretreated with glibenclamide. Combined treatment with GF109203X and mepivacaine enhanced levcromakalim-induced vasodilation compared with pretreatment with mepivacaine alone. This vasodilation was attenuated in aortas precontracted with PDBu compared with those precontracted with phenylephrine. Mepivacaine and glibenclamide, alone or in combination, attenuated levcromakalim-induced membrane hyperpolarization. Taken together, these results suggest that mepivacaine attenuates vasodilation induced by KATP channels, which appears to be partly mediated by PKC.

  11. Glyburide, a K(+)ATP channel blocker, improves hypotension and survival in anaphylactic shock induced in Wistar rats sensitized to ovalbumin.

    PubMed

    Dhanasekaran, Subramanian; Nemmar, Abderrahim; Aburawi, Elhadi H; Kazzam, Elsadig E; Abdulle, Abdishakur; Bellou, Moufida; Bellou, Abdelouahab

    2013-10-25

    Allergens can induce anaphylactic shock and death due to severe hypotension. Potassium channel blockers (K(+)ATP) such as glyburide (GLY) induce vasoconstriction. The effect of K(+)ATP channel blockers on anaphylactic shock is poorly understood. Objective of the study was to test the hypothesis that GLY reduces hypotension induced in anaphylactic shock and increases survival. Rats were grouped into: G1-N=Naïve; G2-SC=Sensitized-Control; G3-SG=Sensitized-GLY (glyburide 40mg/kg); G4-SE=Sensitized-EPI (epinephrine 10µg/kg). G2 to G4 groups were sensitized with ovalbumin (OVA) and shock was induced by i.v. injection of OVA. Treatments were administered intravenously 5min later. Mean arterial pressure (MAP), heart rate (HR), and mean survival time (MST) were measured for 60min following OVA injection and treatments administration. At the end of the experiment, blood withdrawal was performed to measure plasma levels of histamine, leukotriene B4 (LTB4), prostaglandin E2 (PGE2) and prostaglandin F2 (PGF2). Additionally blood gas (paO2, paCO2, SaO2) and electrolytes (Na(+), K(+) and Ca(++)) were measured. MAP was normal in G1-N; severe hypotension, negative inotropic and short MST were observed in G2(-)SC; normalization of MAP, with lesser negative inotropism and increased MST were observed in G3(-)SG; full recovery was observed in G4-SE. Histamine level was significantly higher in G2-SC; reduced in G3-SG and G4-SE. PGE2 increased in G3(-)SG; PGF2 increased in G2-SC and G3(-)SG. Na(+) and Ca(++) concentration decreased in sensitized rats but reversed in treated groups, without change in K(+) concentration. In conclusion, our data suggest that administration of GLY reduces hypotension and increases survival time in rat anaphylactic shock. © 2013 Published by Elsevier B.V.

  12. Characterization of the G protein coupling of a somatostatin receptor to the K+ATP channel in insulin-secreting mammalian HIT and RIN cell lines.

    PubMed Central

    Ribalet, B; Eddlestone, G T

    1995-01-01

    1. The G protein-mediated coupling of a somatostatin (somatotropin-releasing inhibitory factor; SRIF) receptor to the ATP-dependent K+ channel (K+ATP channel) has been studied in insulin-secreting cells using the patch clamp technique. 2. In excised outside-out patches, the concentration-dependent stimulation of the K+ATP channel by SRIF was biphasic. Stimulation reached a maximum at 15 nM (EC50 = 5.5 nM), then decayed to a minimum at 50 nM and returned to maximum stimulation at 500 nM. 3. In cell-attached patches, bath-applied SRIF caused K+ATP channel stimulation in most experiments. In a few cases, however, SRIF suppressed channel activity, a response that was reversed by addition of dibutyryl cyclic AMP (DBcAMP). Channel stimulation by SRIF or by DBcAMP did not occur in the presence of glucose. 4. In excised inside-out patches, the alpha-subunits of Gi or G(o)-type G proteins stimulated the K+ATP channel (EC50 = 29 and 42 pM, respectively). The K+ATP channel stimulation by alpha i- or alpha o-subunits had no effect on the concentration-dependent inhibition by ATP. 5. In excised inside-out patches, K+ATP channel activity was reduced by inhibitors of protein kinase C (PKC) and stimulated by a PKC activator. The stimulatory effect of PKC was unaffected by the presence of pertussis toxin, but stimulation by exogenous alpha-subunits of the G protein Gi or G(o) was prevented by PKC inhibitors. 6. From these data we deduce that SRIF can affect K+ATP channel activity directly via a membrane-delimited pathway or indirectly via a pathway requiring diffusible messengers. In the former case, alpha i/alpha o may either enhance PLC activity, stimulating PKC and thus inducing K+ATP channel phosphorylation with consequent increase of activity, or channel phosphorylation by PKC may facilitate a direct stimulation of the channel by alpha i/alpha o. In the latter case, an alpha i/alpha o-induced fall in cAMP contributes to reduced PKA-mediated phosphorylation and suppression of

  13. 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.

  14. Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP.

    PubMed

    Jin, Jun-Yup; Park, Sung-Hee; Bae, Jae-Hoon; Cho, Ho-Chan; Lim, Jeong-Geun; Park, Won Sun; Han, Jin; Lee, Jin Ho; Song, Dae-Kyu

    2007-09-01

    Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel.

  15. Mitochondrial KATP Channels Control Glioma Radioresistance by Regulating ROS-Induced ERK Activation.

    PubMed

    Huang, Lianyan; Li, Boxing; Tang, Shihao; Guo, Hongbo; Li, Wenjun; Huang, Xiaozhou; Yan, Wenjuan; Zou, Fei

    2015-08-01

    Malignant glioma is the most prevalent form of malignant brain tumor. Although radiotherapy is widely used in glioma treatment, the radioresistance of glioma cells limits the success of the glioma treatment. The lack of effective targets and signaling pathways to reverse glioma radioresistance is the critical obstacle in successful treatment. In this study, we demonstrate that mitochondrial ATP-sensitive potassium channels (mtK(ATP) channels) are overexpressed in glioma cells and are closely related to the malignancy grade and the overall survival of the patients. Importantly, we showed that mtK(ATP) channels could control glioma radioresistance by regulating reactive oxygen species (ROS)-induced ERK activation. The inhibition of mtK(ATP) channels suppresses glioma radioresistance by inhibiting ERK activation both in vitro and in vivo. These findings reveal the important roles of the mitochondria and mtK(ATP) channels as key regulators in the radioresistance of glioma cells, and suggest that mtK(ATP) channel blockers and MAPK/ERK kinase (MEK) inhibitors are potential targets for drug development of glioma treatments.

  16. KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation

    PubMed Central

    Virgili, Noemí; Mancera, Pilar; Wappenhans, Blanca; Sorrosal, Georgina; Biber, Knut

    2013-01-01

    Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer’s disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis. PMID:24040400

  17. 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

  18. Effect of englitazone on KATP and calcium-activated non-selective cation channels in CRI-G1 insulin-secreting cells.

    PubMed

    Rowe, I C; Lee, K; Khan, R N; Ashford, M L

    1997-06-01

    1. The effects of englitazone sodium, an antidiabetic agent, on ion channel activity in the CRI-G1 insulin secreting cell line was examined by use of the patch clamp technique. 2. Application of englitazone to the outside of CRI-G1 cells in the whole-cell recording configuration produced concentration-dependent inhibition of KATP currents with an IC50 value of 8 microM. The inhibition of the K+ current was not affected by the removal of Mg2+ ions from or the addition of trypsin to the solution bathing the intracellular surface of the cell membrane. 3. Englitazone also inhibited KATP channel activity in recordings from inside out excise membrane patches. The concentration-dependence of inhibition was identical to that observed in whole-cell recordings and was voltage-independent. Single channel recordings confirmed that neither the absence or presence of Mg2+ ions nor the addition of trypsin at the intracellular surface of the membrane influenced the inhibition of KATP channels by englitazone. 4. Englitazone also inhibited Ca(2+)-activated non-selective cation (NSCa) channels in inside-out patches in a concentration-dependent and voltage-independent manner with an IC50 value of 10 microM. In comparison, the non-sulphonylurea KATP channel blocker ciclazindol produced a slight voltage-dependent inhibition of the NSCa channel at a concentration of 20 microM. 5. In whole-cell recordings englitazone, at a relatively high concentration (50 microM) in comparison with that required to block KATP and NSCa channels, inhibited voltage-activated Ca2+ currents by 33% but did not inhibit voltage-activated K+ and Na+ currents. 6. It is concluded that englitazone is a novel blocker of NSCa and KATP channels. The inhibition of KATP channels occurs following procedures that dissociate sulphonylurea receptor coupling to the channel. The equipotent and voltage-independent inhibition of NSCa and KATP channels by englitazone may indicate a common mechanism of block.

  19. 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

  20. The N-terminal transmembrane domain (TMD0) and a cytosolic linker (L0) of sulphonylurea receptor define the unique intrinsic gating of KATP channels

    PubMed Central

    Fang, Kun; Csanády, László; Chan, Kim W

    2006-01-01

    ATP-sensitive potassium (KATP) channels comprise four pore-forming Kir6 and four regulatory sulphonylurea receptor (SUR) subunits. SUR, an ATP-binding cassette protein, associates with Kir6 through its N-terminal transmembrane domain (TMD0). TMD0 connects to the core domain of SUR through a cytosolic linker (L0). The intrinsic gating of Kir6.2 is greatly altered by SUR. It has been hypothesized that these changes are conferred by TMD0. Exploiting the fact that the pancreatic (SUR1/Kir6.2) and the cardiac (SUR2A/Kir6.2) KATP channels show different gating behaviours, we have tested this hypothesis by comparing the intrinsic gating of Kir6.2 with the last 26 residues deleted (Kir6.2Δ26) co-expressed with SUR1, S1-TMD0, SUR2A and S2-TMD0 at −40 and −100 mV (S is an abbreviation for SUR; TMD0/Kir6.2Δ26, but not TMD0/Kir6.2, can exit the endoplastic reticulum and reach the cell membrane). Single-channel kinetic analyses revealed that the mean burst and interburst durations are shorter for TMD0/Kir6.2Δ26 than for the corresponding SUR channels. No differences were found between the two TMD0 channels. We further demonstrated that in isolation even TMD0-L0 (SUR truncated after L0) cannot confer the wild-type intrinsic gating to Kir6.2Δ26 and that swapping L0 (SUR truncated after L0)between SUR1 and SUR2A only partially exchanges their different intrinsic gating. Therefore, in addition to TMD0, L0 and the core domain also participate in determining the intrinsic gating of Kir6.2. However, TMD0 and L0 are responsible for the different gating patterns of full-length SUR1 and SUR2A channels. A kinetic model with one open and four closed states is presented to explain our results in a mechanistic context. PMID:16887879

  1. Defects in beta cell Ca²+ signalling, glucose metabolism and insulin secretion in a murine model of K(ATP) channel-induced neonatal diabetes mellitus.

    PubMed

    Benninger, R K P; Remedi, M S; Head, W S; Ustione, A; Piston, D W; Nichols, C G

    2011-05-01

    Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose

  2. Long-pore Electrostatics in Inward-rectifier Potassium Channels

    PubMed Central

    Robertson, Janice L.; Palmer, Lawrence G.; Roux, Benoît

    2008-01-01

    Inward-rectifier potassium (Kir) channels differ from the canonical K+ channel structure in that they possess a long extended pore (∼85 Å) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a K+ ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 Å, suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kir channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores. PMID:19001143

  3. QUATERNARY STRUCTURE OF KATP CHANNEL SUR2A NUCLEOTIDE BINDING DOMAINS RESOLVED BY SYNCHROTRON RADIATION X-RAY SCATTERING

    PubMed Central

    Park, Sungjo; Terzic, Andre

    2009-01-01

    Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K+ (KATP) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168 × 80 × 37 Å3 and 175 × 81 × 37 Å3 based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein-protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of KATP channel SUR2A regulatory domains. PMID:19919849

  4. Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels.

    PubMed

    Rustenbeck, I; Dickel, C; Herrmann, C; Grimmsmann, T

    1999-04-01

    Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

  5. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in C. elegans

    PubMed Central

    Wojtovich, Andrew P.; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2012-01-01

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K+ channel (mKATP) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mKATP may arise from an inward rectifying K+ channel (Kir). The genetic model organism C. elegans exhibits HP and displays mKATP activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mKATP channel activity in purified mitochondria. These data suggest that the mKATP in C. elegans does not arise from a Kir derived channel. PMID:22281198

  6. 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.

  7. The use of microelectrode array (MEA) to study the protective effects of potassium channel openers on metabolically compromised HL-1 cardiomyocytes.

    PubMed

    Law, J K Y; Yeung, C K; Hofmann, B; Ingebrandt, S; Rudd, J A; Offenhäusser, A; Chan, M

    2009-02-01

    The microelectrode array (MEA) was used to evaluate the cardioprotective effects of adenosine triphosphate sensitive potassium (K(ATP)) channel activation using potassium channel openers (KCOs) on HL-1 cardiomyocytes subjected to acute chemically induced metabolic inhibition. Beat frequency and extracellular action potential (exAP) amplitude were measured in the presence of metabolic inhibitors (sodium azide (NaN(3)) or 2-deoxyglucose (2-DG)) or KCOs (pinacidil (PIN, a cyanoguanidine derivative, activates sarcolemmal K(ATP) channels) or SDZ PCO400 (SDZ, a benzopyran derivative, activates mitochondrial K(ATP) channels)). The protective effects of these KCOs on metabolically inhibited HL-1 cells were subsequently investigated. Signal shapes indicated that NaN(3) and 2-DG reduced the rate of the sodium (Na(+)) influx signal as reflected by a reduction in beat frequency. PIN and SDZ appeared to reduce both rate of depolarization and extent of the Na(+) influx signals. Pre-treating cardiomyocytes with PIN (0.1 mM), but not SDZ, prevented the reduction of beat frequency associated with NaN(3)- or 2-DG-induced metabolic inhibition. The exAP amplitude was not affected by either KCO. The cardioprotective effect of PIN relative to SDZ may be due to the opening of different K(ATP) channels. This metabolic inhibition model on the MEA may provide a stable platform for the study of cardiac pathophysiology in the future.

  8. Novel nucleotide-binding sites in ATP-sensitive potassium channels formed at gating interfaces.

    PubMed

    Dong, Ke; Tang, Lie-Qi; MacGregor, Gordon G; Leng, Qiang; Hebert, Steven C

    2005-04-06

    The coupling of cell metabolism to membrane electrical activity is a vital process that regulates insulin secretion, cardiac and neuronal excitability and the responses of cells to ischemia. ATP-sensitive potassium channels (K(ATP); Kir6.x) are a major part of this metabolic-electrical coupling system and translate metabolic signals such as the ATP:ADP ratio to changes in the open or closed state (gate) of the channel. The localization of the nucleotide-binding site (NBS) on Kir6.x channels and how nucleotide binding gates these K(ATP) channels remain unclear. Here, we use fluorescent nucleotide binding to purified Kir6.x proteins to define the peptide segments forming the NBS on Kir6.x channels and show that unique N- and C-terminal interactions from adjacent subunits are required for high-affinity nucleotide binding. The short N- and C-terminal segments comprising the novel intermolecular NBS are next to helices that likely move with channel opening/closing, suggesting a lock-and-key model for ligand gating.

  9. 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.

  10. 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

  11. Activation of ATP-dependent potassium channels is a trigger but not a mediator of ischaemic preconditioning in pigs

    PubMed Central

    Schulz, Rainer; Gres, Petra; Heusch, Gerd

    2003-01-01

    Activation of ATP-dependent potassium channels (KATP) is involved in ischaemic preconditioning (IP). In isolated buffer-perfused rabbit hearts, activation of mitochondrial KATP – through a generation of free radicals – acted as a trigger rather than a mediator of IP; the isolated buffer-perfused heart preparation, however, favours free radical generation. In contrast, in vivo studies in rats and dogs suggested that activation of KATP acts as a mediator of IP's protection. A detailed analysis on the role of KATP in IP's protection in vivo by varying the time and dose of KATP blocker administration is, however, lacking. In 54 enflurane-anaesthetized pigs, the left anterior descending coronary artery was perfused by an extracorporeal circuit. Infarct size (IS, %, TTC) following 90 min sustained low-flow ischaemia and 120 min reperfusion was 26.6±3.5 (s.e.m.) (n=8). IP with one cycle of 10 min ischaemia and 15 min reperfusion reduced IS to 6.5±2.1 (n=7, P<0.05). Blockade of KATP with glibenclamide (0.5 mg kg−1 i.v., 50 μg min−1 continuous infusion) starting 10 min before or immediately following the preconditioning ischaemia abolished IS reduction by IP (20.7±2.7, n=7 and 21.9±6.6, n=6, respectively) while having no effect on IS per se (22.2±5.2, n=7), supporting a trigger role of KATP in IP. In contrast, starting glibenclamide following the preconditioning ischaemia 10 min prior to the sustained ischaemia did not prevent IS reduction by IP (3.7±2.3, n=6), even when its bolus dose was increased to 1.5 mg kg−1 (26.6±3.8 with IP vs 37.5±2.9 without IP; n=7 and 6 respectively, P<0.05), thereby refuting a mediator role of KATP in IP. In conclusion, activation of KATP in the immediate reperfusion following the preconditioning ischaemia is pivotal for triggering IP. PMID:12746224

  12. Arthropod toxins acting on neuronal potassium channels.

    PubMed

    Jiménez-Vargas, Juana María; Possani, Lourival D; Luna-Ramírez, Karen

    2017-09-20

    Arthropod venoms are a rich mixture of biologically active compounds exerting different physiological actions across diverse phyla and affecting multiple organ systems including the central nervous system. Venom compounds can inhibit or activate ion channels, receptors and transporters with high specificity and affinity providing essential insights into ion channel function. In this review, we focus on arthropod toxins (scorpions, spiders, bees and centipedes) acting on neuronal potassium channels. A brief description of the K(+) channels classification and structure is included and a compendium of neuronal K(+) channels and the arthropod toxins that modify them have been listed. Copyright © 2017. Published by Elsevier Ltd.

  13. 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.

  14. The effect of mitochondrial ATP-sensitive potassium channels on apoptosis of chick embryo cecal cells by Eimeria tenella.

    PubMed

    Yang, Sha-sha; Zheng, Ming-xue; Xu, Huan-cheng; Cui, Xiao-zhen; Zhang, Yan; Zhao, Wen-long; Bai, Rui

    2015-04-01

    The objective of this study was to investigate the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channels on apoptosis induced by Eimeria tenella. At 24, 48, 72, 96 and 120 h after Eimeria tenella infection, TUNEL assays and translation of phosphatidyl serines to the host cell plasma membrane surface showed that diazoxide-treated chick embryo cecal cells underwent less apoptosis (P <0.05), while light microscopy showed that infection rates of treated cells were higher (P <0.01) than untreated cells. Caspase 9 and caspase 3 of infected cells were activated less (P <0.01) in diazoxide-treated cells than untreated cells. These results indicate that opening mitoKATP channels can protect chick embryo cecal cells from mitochondria-dependent apoptosis induced by Eimeria tenella by inhibiting activations of caspase 9 and caspase 3.

  15. Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth.

    PubMed

    Jackson, W F

    2017-01-01

    Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca(2+) channels (VGCC), Ca(2+) influx through VGCC, intracellular Ca(2+), and VSM contraction. Membrane potential also affects release of Ca(2+) from internal stores and the Ca(2+) sensitivity of the contractile machinery such that K(+) channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. VSM cells express multiple isoforms of at least five classes of K(+) channels that contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression, and function of large conductance, Ca(2+)-activated K(+) (BKCa) channels, intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels, multiple isoforms of voltage-gated K(+) (KV) channels, ATP-sensitive K(+) (KATP) channels, and inward-rectifier K(+) (KIR) channels in both contractile and proliferating VSM cells. © 2017 Elsevier Inc. All rights reserved.

  16. NMDA receptors mediate leptin signaling and regulate potassium channel trafficking in pancreatic β-cells.

    PubMed

    Wu, Yi; Fortin, Dale A; Cochrane, Veronica A; Chen, Pei-Chun; Shyng, Show-Ling

    2017-08-02

    NMDA receptors (NMDARs) are Ca2+-permeant, ligand-gated ion channels activated by the excitatory neurotransmitter glutamate and have well-characterized roles in the nervous system. The expression and function of NMDARs in pancreatic β cells, by contrast, are poorly understood. Here, we report a novel function of NMDARs in β-cells. Using a combination of biochemistry, electrophysiology, and imaging techniques we now show that NMDARs have a key role in mediating the effect of leptin to modulate β-cell electrical activity by promoting AMP-activated protein kinase (AMPK)-dependent trafficking of KATP and Kv2.1 channels to the plasma membrane. Blocking NMDAR activity inhibited the ability of leptin to activate AMPK, induce KATP and Kv2.1 channel trafficking, and promote membrane hyperpolarization. Conversely, activation of NMDARs mimicked the effect of leptin, causing Ca2+ influx, AMPK activation, increased trafficking of KATP and Kv2.1 channels to the plasma membrane, and triggered membrane hyperpolarization. Moreover, leptin potentiated NMDAR currents and triggered NMDAR-dependent Ca2+ influx. Importantly, NMDAR-mediated signaling was observed in rat insulinoma 832/13 cells and in human β-cells indicating that this pathway is conserved across species. The ability of NMDARs to regulate potassium channel surface expression and thus, β-cell excitability provides mechanistic insight into the recently reported insulinotropic effects of NMDAR antagonists, and therefore highlights the therapeutic potential of these drugs in managing type 2 diabetes. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  17. Significance of KATP channels, L-type Ca2+ channels and CYP450-4A enzymes in oxygen sensing in mouse cremaster muscle arterioles In vivo

    PubMed Central

    2013-01-01

    Background ATP-sensitive K+ channels (KATP channels), NO, prostaglandins, 20-HETE and L-type Ca2+ channels have all been suggested to be involved in oxygen sensing in skeletal muscle arterioles, but the role of the individual mechanisms remain controversial. We aimed to establish the importance of these mechanisms for oxygen sensing in arterioles in an in vivo model of metabolically active skeletal muscle. For this purpose we utilized the exteriorized cremaster muscle of anesthetized mice, in which the cremaster muscle was exposed to controlled perturbation of tissue PO2. Results Change from “high” oxygen tension (PO2 = 153.4 ± 3.4 mmHg) to “low” oxygen tension (PO2 = 13.8 ± 1.3 mmHg) dilated cremaster muscle arterioles from 11.0 ± 0.4 μm to 32.9 ± 0.9 μm (n = 28, P < 0.05). Glibenclamide (KATP channel blocker) caused maximal vasoconstriction, and abolished the dilation to low oxygen, whereas the KATP channel opener cromakalim caused maximal dilation and prevented the constriction to high oxygen. When adding cromakalim on top of glibenclamide or vice versa, the reactivity to oxygen was gradually restored. Inhibition of L-type Ca2+ channels using 3 μM nifedipine did not fully block basal tone in the arterioles, but rendered them unresponsive to changes in PO2. Inhibition of the CYP450-4A enzyme using DDMS blocked vasoconstriction to an increase in PO2, but had no effect on dilation to low PO2. Conclusions We conclude that: 1) L-type Ca2+ channels are central to oxygen sensing, 2) KATP channels are permissive for the arteriolar response to oxygen, but are not directly involved in the oxygen sensing mechanism and 3) CYP450-4A mediated 20-HETE production is involved in vasoconstriction to high PO2. PMID:23663730

  18. Dendritic potassium channels in hippocampal pyramidal neurons

    PubMed Central

    Johnston, Daniel; Hoffman, Dax A; Magee, Jeffrey C; Poolos, Nicholas P; Watanabe, Shigeo; Colbert, Costa M; Migliore, Michele

    2000-01-01

    Potassium channels located in the dendrites of hippocampal CA1 pyramidal neurons control the shape and amplitude of back-propagating action potentials, the amplitude of excitatory postsynaptic potentials and dendritic excitability. Non-uniform gradients in the distribution of potassium channels in the dendrites make the dendritic electrical properties markedly different from those found in the soma. For example, the influence of a fast, calcium-dependent potassium current on action potential repolarization is progressively reduced in the first 150 μm of the apical dendrites, so that action potentials recorded farther than 200 μm from the soma have no fast after-hyperpolarization and are wider than those in the soma. The peak amplitude of back-propagating action potentials is also progressively reduced in the dendrites because of the increasing density of a transient potassium channel with distance from the soma. The activation of this channel can be reduced by the activity of a number of protein kinases as well as by prior depolarization. The depolarization from excitatory postsynaptic potentials (EPSPs) can inactivate these A-type K+ channels and thus lead to an increase in the amplitude of dendritic action potentials, provided the EPSP and the action potentials occur within the appropriate time window. This time window could be in the order of 15 ms and may play a role in long-term potentiation induced by pairing EPSPs and back-propagating action potentials. PMID:10811726

  19. Dendritic potassium channels in hippocampal pyramidal neurons.

    PubMed

    Johnston, D; Hoffman, D A; Magee, J C; Poolos, N P; Watanabe, S; Colbert, C M; Migliore, M

    2000-05-15

    Potassium channels located in the dendrites of hippocampal CA1 pyramidal neurons control the shape and amplitude of back-propagating action potentials, the amplitude of excitatory postsynaptic potentials and dendritic excitability. Non-uniform gradients in the distribution of potassium channels in the dendrites make the dendritic electrical properties markedly different from those found in the soma. For example, the influence of a fast, calcium-dependent potassium current on action potential repolarization is progressively reduced in the first 150 micrometer of the apical dendrites, so that action potentials recorded farther than 200 micrometer from the soma have no fast after-hyperpolarization and are wider than those in the soma. The peak amplitude of back-propagating action potentials is also progressively reduced in the dendrites because of the increasing density of a transient potassium channel with distance from the soma. The activation of this channel can be reduced by the activity of a number of protein kinases as well as by prior depolarization. The depolarization from excitatory postsynaptic potentials (EPSPs) can inactivate these A-type K+ channels and thus lead to an increase in the amplitude of dendritic action potentials, provided the EPSP and the action potentials occur within the appropriate time window. This time window could be in the order of 15 ms and may play a role in long-term potentiation induced by pairing EPSPs and back-propagating action potentials.

  20. Pharmacology of cardiac potassium channels.

    PubMed

    Tamargo, Juan; Caballero, Ricardo; Gómez, Ricardo; Valenzuela, Carmen; Delpón, Eva

    2004-04-01

    Cardiac K+ channels are membrane-spanning proteins that allow the passive movement of K+ ions across the cell membrane along its electrochemical gradient. They regulate the resting membrane potential, the frequency of pacemaker cells and the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters and hormones and class III antiarrhythmic drugs that prolong the action potential duration (APD) and refractoriness and have been found effective to prevent/suppress cardiac arrhythmias. In the human heart, K+ channels include voltage-gated channels, such as the rapidly activating and inactivating transient outward current (Ito1), the ultrarapid (IKur), rapid (IKr) and slow (IKs) components of the delayed rectifier current and the inward rectifier current (IK1), the ligand-gated channels, including the adenosine triphosphate-sensitive (IKATP) and the acetylcholine-activated (IKAch) currents and the leak channels. Changes in the expression of K+ channels explain the regional variations in the morphology and duration of the cardiac action potential among different cardiac regions and are influenced by heart rate, intracellular signalling pathways, drugs and cardiovascular disorders. A progressive number of cardiac and noncardiac drugs block cardiac K+ channels and can cause a marked prolongation of the action potential duration (i.e. an acquired long QT syndrome, LQTS) and a distinct polymorphic ventricular tachycardia termed torsades de pointes. In addition, mutations in the genes encoding IKr (KCNH2/KCNE2) and IKs (KCNQ1/KCNE1) channels have been identified in some types of the congenital long QT syndrome. This review concentrates on the function, molecular determinants, regulation and, particularly, on the mechanism of action of drugs modulating the K+ channels present in the sarcolemma of human cardiac myocytes that contribute to the different phases of the cardiac action

  1. Potassium Channels in Neurofbromatosis-1

    DTIC Science & Technology

    2006-01-01

    Neurofibromatosis-1 (NF-1) is an autosomal dominant genetic disorder commonly associated with cognitive impairments, including low IQ, learning ... disabilities , behavioral difficulties, executive dysfunction and language-based deficits. Despite the growing recognition of the importance of SK channels

  2. Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment

    PubMed Central

    Selivanov, Vitaliy A.; Alekseev, Alexey E.; Hodgson, Denice M.; Dzeja, Petras P.; Terzic, Andre

    2009-01-01

    Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+(KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes. PMID:14977185

  3. Cardiovascular pleiotropic effects of statins and new onset diabetes: is there a common link: do we need to evaluate the role of KATP channels?

    PubMed

    Sehra, Devindra; Sehra, Sudhish; Sehra, Shiv Tej

    2017-07-01

    Statins are considered the main stay of treatment in the prevention of cardio-vascular morbidity and mortality. They have multiple pleiotropic effects, like stabilization of atherosclerotic plaques, inhibition of platelet aggregation, and vascular smooth muscle proliferation; in addition to their lipid lowering action. Statins manifest these pleiotropic effects because they activate KATP channels in the cardiac and vascular tissue. Simultaneous activation of the KATP channels by statins in β cells of pancreas may inhibit insulin release which may lead to diabetes. Areas covered: Literature published between 1980 and 2016 on cholesterol biosynthesis, new onset diabetes and on the pleiotropic effects of statins, was reviewed. A comprehensive search on PubMed, Embase and Cochrane databases was carried out. Expert opinion: Statins exert their beneficial pleiotropic effects on the cardiovascular system by activating KATP channels in the cardiac and vascular tissue. However, simultaneous activation of KATP channels in the beta cells of pancreas leads to inhibition of insulin release. This disturbs the carbohydrate metabolism and probably leads to diabetes. In our opinion, use of stains should be more judicious and restricted to secondary prevention only.

  4. Anti-ischemic properties of a new spiro-cyclic benzopyran activator of the cardiac mito-KATP channel.

    PubMed

    Calderone, Vincenzo; Testai, Lara; Martelli, Alma; Rapposelli, Simona; Digiacomo, Maria; Balsamo, Aldo; Breschi, Maria C

    2010-01-01

    Many activators of K(ATP) channels exhibit cardioprotective effects, mainly mediated by channels expressed on mitochondria (mito-K(ATP)). Previous results showed anti-ischemic effects of the spiro-cyclic derivative A, on isolated rat hearts. In this work this molecule was more extensively studied and diazoxide was used as reference mito-K(ATP) opener. The studies were performed on an in vivo rat model of myocardial infarct and on heart-derived H9c2 cells exposed to an anoxic environment. The mechanism of action was further investigated on isolated rat heart mitochondria. In the model of myocardial infarct compound A and diazoxide produced significant cardioprotective effects, antagonised by the selective mito-K(ATP) blocker 5-hydroxydecanoic acid (5-HD). Compound A, like diazoxide, produced modest and non-significant hypotensive responses, while the hyperglycaemic effects of diazoxide were not observed for the new compound. Protective effects of compound A and diazoxide were also recorded in H9c2 cells and again were inhibited by 5-HD. Compound A and diazoxide caused swelling of cardiac mitochondria, in agreement with the profile of mito-K(ATP) openers. Both compounds evoked concentration-dependent Ca2+-release from Ca2+-preloaded mitochondria, prevented mitochondrial Ca2+-uptake and caused mitochondrial membrane depolarisation. These effects were antagonised by ATP, the endogenous K(ATP) inhibitor. In conclusion, compound A exhibits a promising profile of an anti-ischemic agent, with a mechanism likely to be linked to the activation of mito-K(ATP) channels, and, because of its chemical characteristics such as structural rigidity and chirality due to the spiro-cyclic moiety, represents an interesting template for development of analogues further improved in activity and selectivity.

  5. Sevoflurane postconditioning affects post-ischaemic myocardial mitochondrial ATP-sensitive potassium channel function and apoptosis in ageing rats.

    PubMed

    Jiang, Jing-Jing; Li, Chao; Li, Heng; Zhang, Lei; Lin, Zong-Hang; Fu, Bao-Jun; Zeng, Yin-Ming

    2016-05-01

    This study investigated the effect of sevoflurane postconditioning on post-ischaemic cardiac function, infarct size, myocardial mitochondrial ATP-sensitive potassium channel (mitoKATP) function and apoptosis in ageing rats to determine the possible mechanism underlying the cardioprotective property of sevoflurane. Ageing rat hearts were isolated and attached to a Langendorff apparatus. The hearts were then exposed or not to sevoflurane postconditioning in the presence or absence of 100 μmol/L 5-hydroxydecanoate (5-HD), a selective mitoKATP inhibitor. The infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Mitochondrial morphology was observed by electron microscopy and scored using FlaMeng semiquantitative analysis. In addition, the expression levels of Bax, Bcl-2, and cytochrome-C (Cyt-C) were determined by Western blot analysis at the end of reperfusion. Sevoflurane postconditioning increased coronary flow, improved functional recovery, reduced Bax/Bcl-2 and Cyt-C phosphorylation levels, and decreased mitochondrial lesion severity and the extent of apoptosis. The protective effects of sevoflurane postconditioning were prevented by the mitoKATP inhibitor 5-HD. Sevoflurane postconditioning significantly protected the function of ageing hearts that were subjected to ischaemia and reperfusion, and these protective effects were mediated by mitoKATP opening.

  6. A dual action of saturated fatty acids on electrical activity in rat pancreatic β-cells. Role of volume-regulated anion channel and KATP channel currents.

    PubMed

    Best, L; Jarman, E; Brown, P D

    2011-03-15

    Free fatty acids (FFAs) exert complex actions on pancreatic β-cells. Typically, an initial potentiation of insulin release is followed by a gradual impairment of β-cell function, the latter effect being of possible relevance to hyperlipidaemia in type 2 diabetes mellitus. The molecular actions of FFAs are poorly understood. The present study investigated the acute effects of saturated FFAs on electrophysiological responses of rat pancreatic β-cells. Membrane potential and KATP channel activity were recorded using the perforated patch technique. Volume-regulated anion channel (VRAC) activity was assessed from conventional whole-cell recordings. Cell volume regulation was measured using a video-imaging technique. Addition of octanoate caused a transient potentiation of glucose-induced electrical activity, followed by a gradual hyper-polarisation and a prolonged inhibition of electrical activity. Octanoate caused an initial increase in VRAC activity followed by a secondary inhibition coinciding with increased KATP channel activity. Similar effects were observed with palmitate and 2-bromopalmitate whereas butyrate was virtually ineffective. Octanoate and palmitate also exerted a dual effect on electrical activity evoked by tolbutamide. Octanoate significantly attenuated cell volume regulation in hypotonic solutions, consistent with VRAC inhibition. It is concluded that medium and long chain FFAs have a dual action on glucose-induced electrical activity in rat pancreatic β-cells: an initial stimulatory effect followed by a secondary inhibition. These effects appear to be the result of reciprocal actions on VRAC and KATP channel currents, and could contribute towards the stimulatory and inhibitory actions of FFAs on pancreatic β-cell function.

  7. A dual action of saturated fatty acids on electrical activity in rat pancreatic β-cells. Role of volume-regulated anion channel and KATP channel currents

    PubMed Central

    Best, L; Jarman, E; Brown, P D

    2011-01-01

    Abstract Free fatty acids (FFAs) exert complex actions on pancreatic β-cells. Typically, an initial potentiation of insulin release is followed by a gradual impairment of β-cell function, the latter effect being of possible relevance to hyperlipidaemia in type 2 diabetes mellitus. The molecular actions of FFAs are poorly understood. The present study investigated the acute effects of saturated FFAs on electrophysiological responses of rat pancreatic β-cells. Membrane potential and KATP channel activity were recorded using the perforated patch technique. Volume-regulated anion channel (VRAC) activity was assessed from conventional whole-cell recordings. Cell volume regulation was measured using a video-imaging technique. Addition of octanoate caused a transient potentiation of glucose-induced electrical activity, followed by a gradual hyperpolarisation and a prolonged inhibition of electrical activity. Octanoate caused an initial increase in VRAC activity followed by a secondary inhibition coinciding with increased KATP channel activity. Similar effects were observed with palmitate and 2-bromopalmitate whereas butyrate was virtually ineffective. Octanoate and palmitate also exerted a dual effect on electrical activity evoked by tolbutamide. Octanoate significantly attenuated cell volume regulation in hypotonic solutions, consistent with VRAC inhibition. It is concluded that medium and long chain FFAs have a dual action on glucose-induced electrical activity in rat pancreatic β-cells: an initial stimulatory effect followed by a secondary inhibition. These effects appear to be the result of reciprocal actions on VRAC and KATP channel currents, and could contribute towards the stimulatory and inhibitory actions of FFAs on pancreatic β-cell function. PMID:21242256

  8. Arecoline improves vascular endothelial function in high fructose-fed rats via increasing cystathionine-γ-lyase expression and activating K(ATP) channels.

    PubMed

    Ling, Hong-yan; Wang, Guang; Zhang, Wei; Li, Xing; Zhou, Shou-hong; Hu, Bi

    2012-08-01

    To investigate the effect of arecoline, a major component of betel nut, on vascular endothelial function in high fructose-fed rats and the potential mechanisms underlying the effect. Male Wistar rats were fed a high-fructose or control diet for 16 weeks. At the beginning of week 13, the rats were injected ip with low (0.5 mg·kg(-1)·d(-1)), medium (1.0 mg·kg(-1)·d(-1)) or high (5.0 mg·kg(-1)·d(-1)) doses of arecoline for 4 weeks. At the termination of the treatments, blood was collected, fasting blood glucose (FBG) and serum insulin (FSI) levels were measured, and insulin sensitivity index (ISI) was calculated. The thoracic aortas were isolated and aortic rings were prepared for studying ACh-induced endothelium-dependent vasorelaxation (EDVR). The mRNA and protein expression of cystathionine-γ-lyase (CSE) in the thoracic aortas was analyzed using RT-PCR and Western blot analysis, respectively. In high fructose-fed rats, the levels of FBG and FSI were remarkably increased, whereas the ISI and the mRNA and protein expression of CSE were significantly decreased. ACh-induced EDVR in the aortic rings from high fructose-fed rats was remarkably reduced. These changes were reversed by treatment with high dose arecoline. Pretreatment of the aortic rings rings from high fructose-fed rats with the CSE inhibitor propargylglycine (10 mmol/L) or the ATP-sensitive potassium (K(ATP)) channel blocker glibenclamide (10 mmol/L) abolished the restoration of ACh-induced EDVR by high dose arecoline. On the contrary, treatment with high dose arecoline significantly impaired ACh-induced EDVR in the aortic rings from control rats, and pretreatment with propargylglycine or glibenclamide did not cause further changes. Arecoline treatment improves ACh-induced EDVR in high fructose-fed rats, and the potential mechanism of action might be associated with increase of CSE expression and activation of K(ATP) channels by arecoline.

  9. Arecoline improves vascular endothelial function in high fructose-fed rats via increasing cystathionine-γ-lyase expression and activating KATP channels

    PubMed Central

    Ling, Hong-yan; Wang, Guang; Zhang, Wei; Li, Xing; Zhou, Shou-hong; Hu, Bi

    2012-01-01

    Aim: To investigate the effect of arecoline, a major component of betel nut, on vascular endothelial function in high fructose-fed rats and the potential mechanisms underlying the effect. Methods: Male Wistar rats were fed a high-fructose or control diet for 16 weeks. At the beginning of week 13, the rats were injected ip with low (0.5 mg·kg−1·d−1), medium (1.0 mg·kg−1·d−1) or high (5.0 mg·kg−1·d−1) doses of arecoline for 4 weeks. At the termination of the treatments, blood was collected, fasting blood glucose (FBG) and serum insulin (FSI) levels were measured, and insulin sensitivity index (ISI) was calculated. The thoracic aortas were isolated and aortic rings were prepared for studying ACh-induced endothelium-dependent vasorelaxation (EDVR). The mRNA and protein expression of cystathionine-γ-lyase (CSE) in the thoracic aortas was analyzed using RT-PCR and Western blot analysis, respectively. Results: In high fructose-fed rats, the levels of FBG and FSI were remarkably increased, whereas the ISI and the mRNA and protein expression of CSE were significantly decreased. ACh-induced EDVR in the aortic rings from high fructose-fed rats was remarkably reduced. These changes were reversed by treatment with high dose arecoline. Pretreatment of the aortic rings rings from high fructose-fed rats with the CSE inhibitor propargylglycine (10 mmol/L) or the ATP-sensitive potassium (KATP) channel blocker glibenclamide (10 mmol/L) abolished the restoration of ACh-induced EDVR by high dose arecoline. On the contrary, treatment with high dose arecoline significantly impaired ACh-induced EDVR in the aortic rings from control rats, and pretreatment with propargylglycine or glibenclamide did not cause further changes. Conclusion: Arecoline treatment improves ACh-induced EDVR in high fructose-fed rats, and the potential mechanism of action might be associated with increase of CSE expression and activation of KATP channels by arecoline. PMID:22820911

  10. Single-channel properties of IKs potassium channels.

    PubMed

    Yang, Y; Sigworth, F J

    1998-12-01

    Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K+, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz-20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very "flickery" single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.

  11. Age-related attenuation of isoflurane preconditioning in human atrial cardiomyocytes: roles for mitochondrial respiration and sarcolemmal adenosine triphosphate-sensitive potassium channel activity.

    PubMed

    Mio, Yasushi; Bienengraeber, Martin W; Marinovic, Jasna; Gutterman, David D; Rakic, Mladen; Bosnjak, Zeljko J; Stadnicka, Anna

    2008-04-01

    Clinical trials suggest that anesthetic-induced preconditioning (APC) produces cardioprotection in humans, but the mechanisms of APC and significance of aging for APC in humans are not well understood. Here, the impact of age on the role of two major effectors of APC, mitochondria and sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels, in preconditioning of the human atrial myocardium were investigated. Right atrial appendages were obtained from adult patients undergoing cardiac surgery and assigned to mid-aged (MA) and old-aged (OA) groups. APC was induced by isoflurane in isolated myocardium and isolated cardiomyocytes. Mitochondrial oxygen consumption measurements, myocyte survival testing, and patch clamp techniques were used to investigate mitochondrial respiratory function and sarcKATP channel activity. After in vitro APC with isoflurane, the respiratory function of isolated mitochondria was better preserved after hypoxia-reoxygenation stress in MA than in OA. In isolated intact myocytes, APC significantly decreased oxidative stress-induced cell death in MA but not in OA, and isoflurane protection from cell death was attenuated by the sarcKATP channel inhibitor HMR-1098. Further, the properties of single sarcKATP channels were similar in MA and OA, and isoflurane sensitivity of pinacidil-activated whole cell KATP current was no different between MA and OA myocytes. Anesthetic-induced preconditioning with isoflurane decreases stress-induced cell death and preserves mitochondrial respiratory function to a greater degree in MA than in OA myocytes; however, sarcKATP channel activity is not differentially affected by isoflurane. Therefore, effectiveness of APC in humans may decrease with advancing age partly because of altered mitochondrial function of myocardial cells.

  12. Eukaliuric diuresis and natriuresis in response to the KATP channel blocker U37883A: micropuncture studies on the tubular site of action.

    PubMed

    Huang, D Y; Osswald, H; Vallon, V

    1999-08-01

    1. Systemic application of U37883A, a blocker of ATP sensitive potassium (KATP) channels, elicits diuresis and natriuresis without significantly altering urinary potassium excretion. 2. To elucidate tubular sites of action upstream to the distal nephron, micropuncture experiments were performed in nephrons with superficial glomeruli of anaesthetized Munich-Wistar-Frömter rats during systemic application of U37883A (1, 5 or 15 mg kg-1 i.v.). 3. The observed eukaliuric diuresis and natriuresis in response to U37883A at 15 mg kg-1 was accompanied by an increase in early distal tubular flow rate (VED) from 10 - 18 nl min(-1) reflecting a reduction in fractional reabsorption of fluid up to this site (FR-fluid) of 13%. The latter proposed an effect on water-permeable segments such as the proximal tubule which could fully account for the observed reduction in fractional reabsorption of Na+ up to the early distal tubule (FR-Na+) of 8% and the increase in early distal tubular Na+ concentration ([Na+]ED) from 35 - 51 mM whereas [K+]ED was left unaltered. 4. In comparison, furosemide (3 mg kg-1 i.v.), which acts in the water-impermeable thick ascending limb, elicited diuresis, natriuresis and kaliuresis which were associated with a fall in FR-Na+ of 10% with no change in FR-fluid, and a rise in [Na+]ED from 42 - 117 mM and [K+]ED from 1.2 - 5.7 mM with no change in VED. 5. Direct late proximal tubular fluid collections confirmed a significant inhibition of fluid reabsorption in proximal convoluted tubule in response to systemic application of U37883A. 6. These findings suggest that the diuretic and natriuretic effect upstream to the distal tubule in response to systemic application of U37883A involves actions on water-permeable segments such as the proximal convoluted tubule.

  13. Original 2-alkylamino-6-halogenoquinazolin-4(3H)-ones and K(ATP) channel activity.

    PubMed

    Somers, F; Ouedraogo, R; Antoine, M H; de Tullio, P; Becker, B; Fontaine, J; Damas, J; Dupont, L; Rigo, B; Delarge, J; Lebrun, P; Pirotte, B

    2001-08-02

    A series of 6-substituted 2-alkylaminoquinazolin-4(3H)-ones structurally related to 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides were synthesized and tested as putative K(ATP) channel openers on isolated pancreatic endocrine tissue as well as on isolated vascular, intestinal, and uterine smooth muscle. Most of the 6-halogeno-2-alkylaminoquinazolin-4(3H)-ones were found to inhibit insulin release from pancreatic B-cells and to exhibit vasorelaxant properties. In contrast to their pyridothiadiazine dioxide isosteres previously described as more active on the endocrine than on the smooth muscle tissue, quinazolinones cannot be considered as tissue selective compounds. Biological investigations, including measurements of (86)Rb, (45)Ca efflux from pancreatic islet cells and measurements of vasodilator potency in rat aortic rings exposed to 30 or 80 mM KCl in the presence or the absence of glibenclamide, were carried out with 6-chloro- and 6-iodo-3-isopropylaminoquinazolin-4(3H)-ones. Such experiments showed that, depending on the tissue, these new compounds did not always express the pharmacological profile of pure K(ATP) channel openers. Analyzed by X-ray crystallography, one example of quinazolinones appeared to adopt a double conformation. This only suggests a partial analogy between the 2-alkylaminoquinazolin-4(3H)-ones and the 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides. In conclusion, the newly synthesized quinazolinones interfere with insulin secretion and smooth muscle contractile activity. Most of the compounds lack tissue selectivity, and further investigations are required to fully elucidate their mechanism(s) of action.

  14. Hydrophobic bile salts inhibit gallbladder smooth muscle function via stimulation of GPBAR1 receptors and activation of KATP channels

    PubMed Central

    Lavoie, Brigitte; Balemba, Onesmo B; Godfrey, Cody; Watson, Conall A; Vassileva, Galya; Corvera, Carlos U; Nelson, Mark T; Mawe, Gary M

    2010-01-01

    Hydrophobic bile salts are thought to contribute to the disruption of gallbladder smooth muscle (GBSM) function that occurs in gallstone disease, but their mechanism of action is unknown. The current study was undertaken to determine how hydrophobic bile salts interact with GBSM, and how they reduce GBSM activity. The effect of hydrophobic bile salts on the activity of GBSM was measured by intracellular recording and calcium imaging using wholemount preparations from guinea pig and mouse gallbladder. RT-PCR and immunohistochemistry were used to evaluate expression of the G protein-coupled bile acid receptor, GPBAR1. Application of tauro-chenodeoxycholate (CDC, 50–100 μm) to in situ GBSM rapidly reduced spontaneous Ca2+ flashes and action potentials, and caused a membrane hyperpolarization. Immunoreactivity and transcript for GPBAR1 were detected in gallbladder muscularis. The GPBAR1 agonist, tauro-lithocholic acid (LCA, 10 μm) mimicked the effect of CDC on GBSM. The actions of LCA were blocked by the protein kinase A (PKA) inhibitor, KT5720 (0.5–1.0 μm) and the KATP channel blocker, glibenclamide (10 μm). Furthermore, LCA failed to disrupt GBSM activity in Gpbar1−/− mice. The findings of this study indicate that hydrophobic bile salts activate GPBAR1 on GBSM, and this leads to activation of the cyclic AMP–PKA pathway, and ultimately the opening of KATP channels, thus hyperpolarizing the membrane and decreasing GBSM activity. This inhibitory effect of hydrophobic bile salt activation of GPBAR1 could be a contributing factor in the manifestation of gallstone disease. PMID:20624794

  15. Activation of ATP-dependent potassium channels is a trigger but not a mediator of ischaemic preconditioning in pigs.

    PubMed

    Schulz, Rainer; Gres, Petra; Heusch, Gerd

    2003-05-01

    1. Activation of ATP-dependent potassium channels (K(ATP)) is involved in ischaemic preconditioning (IP). In isolated buffer-perfused rabbit hearts, activation of mitochondrial K(ATP)--through a generation of free radicals--acted as a trigger rather than a mediator of IP; the isolated buffer-perfused heart preparation, however, favours free radical generation. In contrast, in vivo studies in rats and dogs suggested that activation of K(ATP) acts as a mediator of IP's protection. A detailed analysis on the role of K(ATP) in IP's protection in vivo by varying the time and dose of K(ATP) blocker administration is, however, lacking. 2. In 54 enflurane-anaesthetized pigs, the left anterior descending coronary artery was perfused by an extracorporeal circuit. Infarct size (IS, %, TTC) following 90 min sustained low-flow ischaemia and 120 min reperfusion was 26.6+/-3.5 (s.e.m.) (n=8). IP with one cycle of 10 min ischaemia and 15 min reperfusion reduced IS to 6.5+/-2.1 (n=7, P<0.05). Blockade of K(ATP) with glibenclamide (0.5 mg kg(-1) i.v., 50 microg min(-1) continuous infusion) starting 10 min before or immediately following the preconditioning ischaemia abolished IS reduction by IP (20.7+/-2.7, n=7 and 21.9+/-6.6, n=6, respectively) while having no effect on IS per se (22.2+/-5.2, n=7), supporting a trigger role of K(ATP) in IP. In contrast, starting glibenclamide following the preconditioning ischaemia 10 min prior to the sustained ischaemia did not prevent IS reduction by IP (3.7+/-2.3, n=6), even when its bolus dose was increased to 1.5 mg kg(-1) (26.6+/-3.8 with IP vs 37.5+/-2.9 without IP; n=7 and 6 respectively, P<0.05), thereby refuting a mediator role of K(ATP) in IP. 3. In conclusion, activation of K(ATP) in the immediate reperfusion following the preconditioning ischaemia is pivotal for triggering IP.

  16. Mutations of the Same Conserved Glutamate Residue in NBD2 of the Sulfonylurea Receptor 1 Subunit of the KATP Channel Can Result in Either Hyperinsulinism or Neonatal Diabetes

    PubMed Central

    Männikkö, Roope; Flanagan, Sarah E.; Sim, Xiuli; Segal, David; Hussain, Khalid; Ellard, Sian; Hattersley, Andrew T.; Ashcroft, Frances M.

    2011-01-01

    OBJECTIVE Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K+ channel (KATP channel) sulfonylurea receptor 1 (SUR1) subunit were detected heterozygously in patients with neonatal diabetes. A mutation at the same residue (E1506K) was previously shown to cause congenital hyperinsulinemia. We sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia. RESEARCH DESIGN AND METHODS Neonatal diabetic patients were sequenced for mutations in ABCC8 (SUR1) and KCNJ11 (Kir6.2). Wild-type and mutant KATP channels were expressed in Xenopus laevis oocytes and studied with electrophysiological methods. RESULTS Oocytes expressing neonatal diabetes mutant channels had larger resting whole-cell KATP currents than wild-type, consistent with the patients’ diabetes. Conversely, no E1506K currents were recorded at rest or after metabolic inhibition, as expected for a mutation causing hyperinsulinemia. KATP channels are activated by Mg-nucleotides (via SUR1) and blocked by ATP (via Kir6.2). All mutations decreased channel activation by MgADP but had little effect on MgATP activation, as assessed using an ATP-insensitive Kir6.2 subunit. Importantly, using wild-type Kir6.2, a 30-s preconditioning exposure to physiological MgATP concentrations (>300 µmol/L) caused a marked reduction in the ATP sensitivity of neonatal diabetic channels, a small decrease in that of wild-type channels, and no change for E1506K channels. This difference in MgATP inhibition may explain the difference in resting whole-cell currents found for the neonatal diabetes and hyperinsulinemia mutations. CONCLUSIONS Mutations in the same residue can cause either hyperinsulinemia or neonatal diabetes. Differentially altered nucleotide regulation by NBD2 of SUR1 can explain the respective clinical phenotypes. PMID:21617188

  17. Large-conductance Ca2+-activated potassium channels in secretory neurons.

    PubMed

    Lara, J; Acevedo, J J; Onetti, C G

    1999-09-01

    Large-conductance Ca2+-activated K+ channels (BK) are believed to underlie interburst intervals and contribute to the control of hormone release in several secretory cells. In crustacean neurosecretory cells, Ca2+ entry associated with electrical activity could act as a modulator of membrane K+ conductance. Therefore we studied the contribution of BK channels to the macroscopic outward current in the X-organ of crayfish, and their participation in electrophysiological activity, as well as their sensitivity toward intracellular Ca2+, ATP, and voltage, by using the patch-clamp technique. The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to K+ ions; potassium permeability (PK) value was 2.3 x 10(-13) cm(3) s(-1). The BK channels were sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP. Voltage sensitivity (k) was approximately 13 mV, and the half-activation membrane potentials depended on the internal Ca2+ concentration. Calcium ions (0.3-3 microM) applied to the internal membrane surface caused an enhancement of the channel activity. This activation of BK channels by internal calcium had a KD(0) of 0.22 microM and was probably due to the binding of only one or two Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to the internal solution increased steady state-open probability. The dissociation constant for MgATP (KD) was 119 microM, and the Hill coefficient (h) was 0.6, according to the Hill analysis. Ca2+-activated K+ currents recorded from whole cells were suppressed by either adding Cd2+ (0.4 mM) or removing Ca2+ ions from the external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these currents. Our results showed that both BK and K(ATP) channels are present in the same cell. Even when BK and K(ATP) channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite different for each kind

  18. Gating of two pore domain potassium channels

    PubMed Central

    Mathie, Alistair; Al-Moubarak, Ehab; Veale, Emma L

    2010-01-01

    Two-pore-domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K+ channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K+ channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C-type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist. PMID:20566661

  19. Gating of two pore domain potassium channels.

    PubMed

    Mathie, Alistair; Al-Moubarak, Ehab; Veale, Emma L

    2010-09-01

    Two-pore-domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K(+) channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K(+) channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C-type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist.

  20. Nicorandil prevents oxidative stress-induced apoptosis in neurons by activating mitochondrial ATP-sensitive potassium channels.

    PubMed

    Teshima, Yasushi; Akao, Masaharu; Baumgartner, William A; Marbán, Eduardo

    2003-11-14

    Nicorandil, a clinically useful drug for the treatment of ischemic heart disease, has an anti-apoptotic effect in cardiomyocytes, and activation of mitochondrial ATP-sensitive potassium (mitoKATP) channels underlies this effect. Recently, several studies showed that nicorandil reduced brain injury in animal models of brain ischemia. Based on these facts, we hypothesized that nicorandil may have anti-apoptotic effects in neurons mediated by mitoKATP channels. We investigated the effect of nicorandil on apoptosis induced by oxidative stress using cultured cerebellar granule neurons. Nicorandil (100 micromol/l) significantly suppressed the number of cells with TUNEL-positive nuclei and the increase in caspase-3 activity induced by 20 micromol/l H2O2. An indicator dye for mitochondrial inner membrane potential (DeltaPsim) revealed that nicorandil prevented the loss of DeltaPsim induced by H2O2 in a concentration-dependent manner. These effects were abolished by 5-hydroxydecanoate (5HD; 500 micromol/l), a mitoKATP channel blocker. The present results showed that nicorandil has anti-apoptotic effects in neurons, at least in part, by preserving DeltaPsim.

  1. Active dendrites, potassium channels and synaptic plasticity.

    PubMed Central

    Johnston, Daniel; Christie, Brian R; Frick, Andreas; Gray, Richard; Hoffman, Dax A; Schexnayder, Lalania K; Watanabe, Shigeo; Yuan, Li-Lian

    2003-01-01

    The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons. PMID:12740112

  2. Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

    PubMed Central

    de Paoli, Frank Vincenzo; Flatman, John A.; Nielsen, Ole Bækgaard

    2009-01-01

    Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (Gm), which in skeletal muscle fibers covers membrane conductances for K+ (GK) and Cl− (GCl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of GK and GCl to inversely interfere with muscle excitability. However, whether regulation of GK and GCl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of GCl and GK with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of Gm in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in GCl of ∼70% caused Gm to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in GK (∼14-fold) and GCl (∼3-fold) caused Gm to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, Gm recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (KATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl− channel inhibitor) revealed that the decreased Gm during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated Gm during phase 2 reflected synchronized openings of ClC-1 and KATP channels. In conclusion, GCl and GK are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle. PMID

  3. Regulation of ClC-1 and KATP channels in action potential-firing fast-twitch muscle fibers.

    PubMed

    Pedersen, Thomas Holm; de Paoli, Frank Vincenzo; de Paoli, Frank Vinzenco; Flatman, John A; Nielsen, Ole Baekgaard

    2009-10-01

    Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G(m)), which in skeletal muscle fibers covers membrane conductances for K(+) (G(K)) and Cl(-) (G(Cl)). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G(K) and G(Cl) to inversely interfere with muscle excitability. However, whether regulation of G(K) and G(Cl) occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G(Cl) and G(K) with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G(m) in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G(Cl) of approximately 70% caused G(m) to decline by approximately 55% in a manner that is well described by a single exponential function characterized by a time constant of approximately 200 APs (phase 1). When stimulation was continued beyond approximately 1,800 APs, synchronized elevations in G(K) ( approximately 14-fold) and G(Cl) ( approximately 3-fold) caused G(m) to rise sigmoidally to approximately 400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G(m) recovered to its level before AP firing in approximately 1 min. Experiments with glibenclamide (K(ATP) channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl(-) channel inhibitor) revealed that the decreased G(m) during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G(m) during phase 2 reflected synchronized openings of ClC-1 and K(ATP) channels. In conclusion, G(Cl) and G(K) are acutely regulated in AP

  4. The influence of animal species on the relationship between ATP-sensitive potassium ion channels and defense reflexes of the airways.

    PubMed

    Sutovska, M; Franova, S; Sutovsky, J

    2009-01-01

    The ATP-sensitive potassium ion channels (K+ATP) are widely expressed on airway sensory nerves that mediate cough and other protective reflexes; dependent on airways smooth muscles (ASM) reactivity. The study was conducted to determine the involvement of K+ATP in airway sensory nerves activity of different animal species. In this study we have evaluated the cough reflex inhibiting potential and modulation of ASM reactivity in vitro, by K+ATP opener-pinacidil in guinea pig and cats. The cough in guinea pig was induced by administration, in cats by mechanical stimulation of the airways. ASM reactivity was tested by organ tissue both method of 0.3 M citric acid aerosol at 3 min interval. 1) Pinacidil inhibits cough reflex in guinea pigs which was antagonized when pre-treated with K+ATP blocker glibenclamide. 2) Pinacidil exhibited antitussive activity in cats comparable to codeine and was noticeably higher than dropropizine effects. 3) ASM reactivity was significantly abolished by pinacidil and almost completely antagonized by glibenclamide in guinea pigs. 4) Pinacidil significantly attenuated ASM contraction in cats only in highest concentrations of contractile mediators. K+ATP may be involved in mechanisms of cough reflex, likewise in antitussive activity of several more agents and also on defence reflexes dependent on ASM reactivity in different animal species such as guinea pigs and cats (Fig. 8, Ref. 20). Full Text (Free, PDF) www.bmj.sk.

  5. The Ketogenic Diet and Potassium Channel Function

    DTIC Science & Technology

    2014-10-01

    1 AWARD NUMBER: W81XWH-13-1-0463 TITLE: The Ketogenic Diet and Potassium Channel Function...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The overall objective of this Discovery Award is to explore the hypothesis the ketogenic diet ...have examining the impact of the ketogenic diet on mice in which the gene that encodes Kvβ2 has been deleted (Kvβ2 KO mice) using an in vitro model of

  6. The Ketogenic Diet and Potassium Channel Function

    DTIC Science & Technology

    2015-11-01

    observation is not reversed by the KD which to some extent supports our initial hypothesis. 15. SUBJECT TERMS Epilepsy , Ketogenic Diet, Seizure Disorder...ketogenic diet (KD), which is used to treat epilepsy (primarily in children) exerts a positive effect on seizure activity by regulating neuronal... Epilepsy , Ketogenic Diet, Seizure Disorder, Potassium Channels, Neurophysiology 3. Overall Project Summary: To determine the impact of KD on

  7. [Discovery of potential ATP-sensitive potassium channel openers with potential hypotensive activity from Chinese herbs based on molecular simulation].

    PubMed

    Li, Yong; Jiang, Lu-di; Chen, Xi; He, Yu-Su; Li, Gong-Yu; Zhang, Yan-Ling

    2016-01-01

    In this research, a combined method of ligand-based pharmacophore (LBP), structure-based pharmacophore (SBP), and molecular docking was applied for virtual screening potential ATP-sensitive potassium channel (KATP) openers from Chinese herbs. LBP models were generated by 3D-QSAR pharmacophore(hypogen) program, based on the training set composed of 48 KATP agonists. The best LBP model consisted of one hydrogen-bond acceptor, one hydrogen-bond donor, one hydrophobic feature, one aromatic ring and five excluded volumes. Besides, the correlation coefficient of training set and test set, N, and CAI value of the model were 0.876 4, 0.705 8, 3.304, and 2.616 respectively. Meanwhile, SBP models were also generated based on a 3D structure of KATP (PMID: PM0079770). The best SBP model consisted of six hydrogen-bond acceptors, eight hydrogen-bond donors, seven hydrophobic features and eighteen excluded volumes. The corresponding N and CAI value were 2.200 and 2.017. Then, the best LBP model and SBP model were applied to identify potential KATP openers from Traditional Chinese Medicine Database(TCMD), respectively. 349 hits were obtained after analyzed by drug-likeness rules. Moreover, 12 compounds with high docking scores were reserved after molecular docking evaluation. Interestingly, part of the results had been verified as hypotensive active ingredients by literatures. Therefore, this study uncovers a specific target effect contained in TCMD, and provides candidates for new KATP openers' research. Copyright© by the Chinese Pharmaceutical Association.

  8. 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.

  9. 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

  10. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

    PubMed Central

    Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung

    2011-01-01

    Background Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue. Methods and Findings Single-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions. Conclusion The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in

  12. 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

  13. [What we don't know about mitochondrial potassium channels?

    PubMed

    Augustynek, Bartłomiej; Wrzosek, Antoni; Koprowski, Piotr; Kiełbasa, Agnieszka; Bednarczyk, Piotr; Łukasiak, Agnieszka; Dołowy, Krzysztof; Szewczyk, Adam

    2016-01-01

    In the current work the authors present the most interesting, yet not fully understood issues regarding origin, function and pharmacology of the mitochondrial potassium channels. There are eight potassium channels known to contribute to the potassium permeability of the inner mitochondrial membrane: ATP-regulated channel, calcium-regulated channels of large, intermediate and small conductance, voltage-regulated Kv1.3 and Kv7.4 channels, two-pore-domain TASK-3 channel and SLO2 channel. The primary function of the mitochondrial potassium channels is regulation of the mitochondrial membrane potential. Additionally, mitochondrial potassium channels alter cellular respiration, regulation of the mitochondrial volume and ROS synthesis. However, mechanisms underlying these processes are not fully understood yet. In this work, the authors not only present available knowledge about this topic, but also put certain hypotheses that may set the direction for the future research on these proteins.

  14. 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

  15. 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

    2015-09-25

    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.

  16. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Acarbose reduces myocardial infarct size by preventing postprandial hyperglycemia and hydroxyl radical production and opening mitochondrial KATP channels in rabbits.

    PubMed

    Minatoguchi, Shinya; Zhang, Zengi; Bao, Narentuoya; Kobayashi, Hiroyuki; Yasuda, Shinji; Iwasa, Masamitsu; Sumi, Syouhei; Kawamura, Itta; Yamada, Yoshihisa; Nishigaki, Kazuhiko; Takemura, Genzou; Fujiwara, Takako; Fujiwara, Hisayoshi

    2009-07-01

    Acarbose, an antidiabetic drug, is an alpha-glucosidase inhibitor that can inhibit glucose absorption in the intestine. A recent large-scale clinical trial, STOP-NIDDM, showed that acarbose reduces the risk of myocardial infarction. We examined whether acarbose reduces myocardial infarct size and investigated its mechanisms. Rabbits were fed with 1 of 2 diets in this study: normal chow, 30 mg acarbose per 100 g chow for 7 days. Rabbits were assigned randomly to 1 of 4 groups: control (n = 10), acarbose (n = 10), acarbose + 5HD (n = 10, intravenous 5 mg/kg of 5-hydroxydecanoate), and 5HD (n = 10, intravenous 5 mg/kg of 5HD). Rabbits then underwent 30 minutes of coronary occlusion followed by 48-hour reperfusion. Postprandial blood glucose levels were higher in the control group than in the acarbose group. The infarct size as a percentage of the left ventricular area at risk was reduced significantly in the acarbose (19.4% +/- 2.3%) compared with the control groups (42.8% +/- 5.4%). The infarct size-reducing effect of acarbose was abolished by 5HD (43.4% +/- 4.7%). Myocardial interstitial 2,5-dihydroxybenzoic acid levels, an indicator of hydroxyl radicals, increased during reperfusion after 30 minutes of ischemia, but this increase was inhibited in the acarbose group. This was reversed by 5HD. Acarbose reduces myocardial infarct size by opening mitochondrial KATP channels, which may be related to the prevention of postprandial hyperglycemia and hydroxyl radical production.

  18. Molecular action of sulphonylureas on KATP channels: a real partnership between drugs and nucleotides

    PubMed Central

    de Wet, Heidi; Proks, Peter

    2015-01-01

    Sulphonylureas stimulate insulin secretion from pancreatic β-cells primarily by closing ATP-sensitive K+ channels in the β-cell plasma membrane. The mechanism of channel inhibition by these drugs is unusually complex. As direct inhibitors of channel activity, sulphonylureas act only as partial antagonists at therapeutic concentrations. However, they also exert an additional indirect inhibitory effect via modulation of nucleotide-dependent channel gating. In this review, we summarize current knowledge and recent advances in our understanding of the molecular mechanism of action of these drugs. PMID:26517901

  19. K(ATP) channel gain-of-function leads to increased myocardial L-type Ca(2+) current and contractility in Cantu syndrome.

    PubMed

    Levin, Mark D; Singh, Gautam K; Zhang, Hai Xia; Uchida, Keita; Kozel, Beth A; Stein, Phyllis K; Kovacs, Atilla; Westenbroek, Ruth E; Catterall, William A; Grange, Dorothy Katherine; Nichols, Colin G

    2016-06-14

    Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca(2+) current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology.

  20. KATP channel gain-of-function leads to increased myocardial L-type Ca2+ current and contractility in Cantu syndrome

    PubMed Central

    Levin, Mark D.; Singh, Gautam K.; Zhang, Hai Xia; Uchida, Keita; Kozel, Beth A.; Stein, Phyllis K.; Kovacs, Atilla; Westenbroek, Ruth E.; Catterall, William A.; Grange, Dorothy Katherine; Nichols, Colin G.

    2016-01-01

    Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca2+ current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology. PMID:27247394

  1. Voltage sensor inactivation in potassium channels.

    PubMed

    Bähring, Robert; Barghaan, Jan; Westermeier, Regina; Wollberg, Jessica

    2012-01-01

    In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive ("P/C-type inactivation"). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as "preferential" closed-state inactivation, we introduce the term "A/C-type inactivation". Mechanistically, P/C- and A/C-type inactivation represent two forms of "voltage sensor inactivation."

  2. 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.

  3. Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures.

    PubMed

    Piwońska, Marta; Szewczyk, Adam; Schröder, Ullrich H; Reymann, Klaus G; Bednarczyk, Iotr

    2016-01-01

    Mitochondria have been suggested as a potential target for cytoprotective strategies. It has been shown that increased K+ uptake mediate by mitochondrial ATP-regulated potassium channels (mitoKATP channel) or large-conductance Ca2+-activated potassium channels (mitoBKCa channel) may provide protection in different models of cell death. Since recent findings demonstrated the presence of BKCa channels in neuronal mitochondria, the goal of the present study was to test the potential neuroprotective effects of BKCa channel modulators. Using organotypic hippocampal slice cultures exposed to glutamate, we demonstrated that preincubation of the slices with the BKCa channel opener NS1619 resulted in decreased neuronal cell death measured as reduced uptake of propidium iodide. This neuroprotective effect was reversed by preincubation with the BKCa channel inhibitors paxilline and Iberiotoxin (IbTx). Moreover, mitochondrial respiration measurements revealed that NS1619 induced an IbTx-sensitive increase in state 2 respiration of isolated brain mitochondria. In addition, electrophysiological patch-clamp studies confirmed the presence of BKCa channels in mitoplasts isolated from embryonic hippocampal cells. Taken together, our results confirm presence of BKCa channel in rat hippocampal neurons mitochondria and suggest putative role for mitoBKCa in neuroprotection.

  4. Metamizol acts as an ATP sensitive potassium channel opener to inhibit the contracting response induced by angiotensin II but not to norepinephrine in rat thoracic aorta smooth muscle.

    PubMed

    Valenzuela, Fermín; García-Saisó, Sebastián; Lemini, Cristina; Ramírez-Solares, Rafael; Vidrio, Horacio; Mendoza-Fernández, Víctor

    2005-08-01

    Clinically metamizol (MZ) has been related to alteration on haemodynamic parameters and modifications on blood pressure in humans when administered intravenously. These effects have been observed at MZ therapeutic doses. Experimentally, MZ is able to induce relaxation on several types of vascular smooth muscles and modulates the contraction induced by phenylephrine. However, the mechanism underlying the MZ effects on vascular reactivity is not clear. Potassium channels (K) present on vascular smooth muscle cells closely regulate the vascular reactivity and membrane potential. There are four described types of K in vascular tissue: K voltage sensitive (K(V)), K calcium sensitive (K(Ca)2+), K ATP sensitive (K(ATP) and K inward rectification (K(IR), voltage sensitive). The aim of this work was to investigate MZ effects on angiotensin II (AT II) and noradrenaline (NA) induced contraction and to evaluate the K participation on MZ modulating effect on vascular smooth muscle contraction, using isometric and patch clamp techniques. MZ induces relaxation in a concentration dependent manner. Furthermore, MZ strongly inhibits in a concentration dependent fashion the contraction induced by AT II. However, MZ inhibition on NA induced contraction was moderated compared with that observed on AT II. MZ effects on AT II induced contraction was blocked by glybenclamide (a specific K(ATP) blocker, 3 microM, *p < 0.01). In patch clamp experiments, MZ (3 mM) induces an increase on potassium current (K+) mediated by K(ATP) in similar way as diazoxide (a specific K(ATP) opener, 3 microM). Our results suggest that MZ induces relaxation and inhibits contraction induced by AT II acting as a K(ATP) opener.

  5. Sevoflurane Preconditioning Reduces Intestinal Ischemia-Reperfusion Injury: Role of Protein Kinase C and Mitochondrial ATP-Sensitive Potassium Channel

    PubMed Central

    Shen, Zhiwen; Miao, Liping; Zhang, Kun; Wang, Fei; Li, Yujuan

    2015-01-01

    Ischemic preconditioning (IPC) has been considered to be a potential therapy to reduce ischemia-reperfusion injury (IRI) since the 1980s. Our previous study indicated that sevoflurane preconditioning (SPC) also reduced intestinal IRI in rats. However, whether the protective effect of SPC is similar to IPC and the mechanisms of SPC are unclear. Thus, we compared the efficacy of SPC and IPC against intestinal IRI and the role of protein kinase C (PKC) and mitochondrial ATP-sensitive potassium channel (mKATP) in SPC. A rat model of intestinal IRI was used in this study. The superior mesenteric artery (SMA) was clamped for 60 min followed by 120 min of reperfusion. Rats with IPC underwent three cycles of SMA occlusion for 5 min and reperfusion for 5 min before intestinal ischemia. Rats with SPC inhaled sevoflurane at 0.5 minimum alveolar concentration (MAC) for 30 min before the intestinal ischemic insult. Additionally, the PKC inhibitor Chelerythrine (CHE) or mKATP inhibitor 5-Hydroxydecanoic (5-HD) was injected intraperitoneally before sevoflurane inhalation. Both SPC and IPC ameliorated intestinal IRI-induced histopathological changes, decreased Chiu’s scores, reduced terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the epithelium, and inhibited the expression of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α). These protective effects of SPC were similar to those of IPC. Pretreatment with PKC or mKATP inhibitor abolished SPC—induced protective effects by increasing Chiu’s scores, down-regulated the expression of Bcl-2 and activated caspase-3. Our results suggest that pretreatment with 0.5 MAC sevoflurane is as effective as IPC against intestinal IRI. The activation of PKC and mKATP may be involved in the protective mechanisms of SPC. PMID:26505750

  6. Cardioprotection from ischemia/reperfusion induced by red wine extract is mediated by K(ATP) channels.

    PubMed

    Mosca, Susana M; Cingolani, Horacio E

    2002-09-01

    The objective was to analyze the mechanism of the protection induced by a nonalcoholic extract of red wine (RWE) on ischemia/reperfusion injury. Isovolumic perfused rat hearts were exposed after stabilization to a 20-min global ischemic period followed by 30 min of reperfusion in absence and presence of RWE infused prior to ischemia and early in reperfusion. In other hearts, 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) blocker, chelerythrine (1 microM), a protein kinase C blocker, or >L(G)-nitro->L-arginine methyl ester (>L-NAME), a nitric oxide synthase inhibitor, was administered prior to RWE infusion. Left ventricular developed pressure (LVDP), +dP/dtmax, and left ventricular end-diastolic pressure (LVEDP) were used to assess myocardial function. The lactate dehydrogenase release during reperfusion was measured. After the ischemic period, LVDP decreased to 61 +/- 4% and +dP/dtmax to 62 +/- 5% of baseline values at the end of reperfusion. The infusion of RWE resulted in a complete recovery of systolic function (LVDP = 102 +/- 4%; +dP/dtmax = 101 +/- 4%) and in an attenuation of the increase of LVEDP (20 +/- 3 mm Hg versus 42 +/- 4 mm Hg, p < 0.05). The treatment with RWE did not produce lactate dehydrogenase release during reperfusion. 5-HD and chelerythrine completely abolished the protection induced by RWE (mechanical and enzymatic). >L-NAME partially abolished the systolic improvement induced by RWE but returned lactate dehydrogenase loss to ischemic control values. The diastolic protection afforded by RWE was not altered by >L-NAME. These data are the first demonstration that mitochondrial K channels and nitric oxide are involved in the protection against ischemia/reperfusion conferred by a nonalcoholic RWE.

  7. Proteinase inhibitor homologues as potassium channel blockers.

    PubMed

    Lancelin, J M; Foray, M F; Poncin, M; Hollecker, M; Marion, D

    1994-04-01

    We report here the NMR structure of dendrotoxin I, a powerful potassium channel blocker from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (black mamba), calculated from an experimentally-derived set of 719 geometric restraints. The backbone of the toxin superimposes on bovine pancreatic trypsin inhibitor (BPTI) with a root-mean-square deviation of < 1.7 A. The surface electrostatic potential calculated for dendrotoxin I and BPTI, reveal an important difference which might account for the differences in function of the two proteins. These proteins may provide examples of adaptation for specific and diverse biological functions while at the same time maintaining the overall three-dimensional structure of a common ancestor.

  8. 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.

  9. 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

  10. Sheng-Mai-San is protective against post-ischemic myocardial dysfunction in rats through its opening of the mitochondrial KATP channels.

    PubMed

    Wang, Ningyuan; Minatoguchi, Shinya; Arai, Masazumi; Uno, Yoshihiro; Nishida, Yoshio; Hashimoto, Kazuaki; Xue-Hai, Chen; Fukuda, Kazunori; Akao, Seigo; Takemura, Genzou; Fujiwara, Hisayoshi

    2002-08-01

    The present study used isolated rat hearts to investigate whether (1) Sheng-Mei-San (SMS), a traditional Chinese formulation comprising Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae, is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effect of SMS is related to scavenging of hydroxyl radicals and opening the mitochondrial KATP channels. The excised hearts of male Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s) and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min consisting of a 30-min pre-ischemic period followed by a 30-min global ischemia and 60-min reperfusion. Lactate, lactate dehydrogenase (LDH) and 2,5-dihydroxybenzoic acid (2,5-DHBA) concentrations in the effluent were measured during reperfusion. Three days' treatment with SMS (1.67 ml/kg per day) inhibited the rise in LVEDP and improved the post-ischemic LVDP and +/-dP/dt significantly better than in the untreated control hearts during reperfusion. SMS increased the coronary flow at baseline, and during reperfusion. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial KATP channel blocker, abolished the inhibition of the rise in LVEDP, the increase in coronary flow and the improvement in LVDP and +/-dP/dt induced by SMS. SMS significantly attenuated the concentrations of lactate, LDH and 2,5-DHBA during reperfusion, but the pretreatment with 5-HD restored them; 5-HD alone did not affect the concentrations. SMS improved the post-ischemic myocardial dysfunction through opening the mitochondrial KATP channels.

  11. 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

  12. Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel.

    PubMed

    Gross, Eric R; Nithipatikom, Kasem; Hsu, Anna K; Peart, Jason N; Falck, John R; Campbell, William B; Gross, Garrett J

    2004-12-01

    Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.

  13. Diazoxide, a K(ATP) channel opener, prevents ischemia-reperfusion injury in rodent pancreatic islets.

    PubMed

    Wang, Yong; Wang, Shusen; Harvat, Tricia; Kinzer, Katie; Zhang, Lisa; Feng, Feng; Qi, Meirigeng; Oberholzer, Jose

    2015-01-01

    Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K(+) channels that has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here we investigated whether diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in a rodent ischemia model. C57/B6 mice pancreata were flushed with UW or UW + DZ solution and cold preserved for 6 or 10 h prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Significantly higher islet yields were observed in the UW + DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW + DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (60.96 ng/islet ± 13.94 vs. 42.09 ng/islet ± 8.15, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials, and calcium influx responded to glucose. A higher number of living cells and less late apoptotic cells were observed in the UW + DZ group (p < 0.05). Additionally, the islets from the UW + DZ group had a significantly higher cure rate and improved glucose tolerance. This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. In the future, it will be necessary to further understand the underlying mechanism for the mitoprotection and to test this promising approach for pancreas preservation and the islet isolation process in nonhuman primates and ultimately humans.

  14. Diazoxide, a KATP Channel Opener, Prevents Ischemia-Reperfusion Injury in Rodent Pancreatic Islets

    PubMed Central

    Wang, Yong; Wang, Shusen; Harvat, Tricia; Kinzer, Katie; Zhang, Lisa; Feng, Feng; Qi, Meirigeng; Oberholzer, Jose

    2014-01-01

    Objective Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K+ channels and has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here, we investigated whether Diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in rodent ischemia model. Research Design and Methods C57/B6 mice pancreata were flushed with UW or UW+DZ solution and cold preserved for 6 or 10 hrs prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Results Significantly higher islet yields were observed in the UW+DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW+DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (6095.6 ng/islet ± 1394.5 vs. 4209.2 ng/islet ± 815.1, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials and calcium influx responded to glucose. Higher living cells and less late apoptotic cells were observed in the UW+DZ group (p < 0.05). Additionally, the transplanted islets from the UW+DZ group had a significantly higher cure rate and improved glucose tolerance. Conclusion This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. It remains to be tested whether these findings can be replicated in human islet isolation and transplantation. PMID:24070013

  15. Mitochondrial large-conductance potassium channel from Dictyostelium discoideum.

    PubMed

    Laskowski, Michal; Kicinska, Anna; Szewczyk, Adam; Jarmuszkiewicz, Wieslawa

    2015-03-01

    In the present study, we describe the existence of a large-conductance calcium-activated potassium (BKCa) channel in the mitochondria of Dictyostelium discoideum. A single-channel current was recorded in a reconstituted system, using planar lipid bilayers. The large-conductance potassium channel activity of 258±12 pS was recorded in a 50/150 mM KCl gradient solution. The probability of channel opening (the channel activity) was increased by calcium ions and NS1619 (potassium channel opener) and reduced by iberiotoxin (BKCa channel inhibitor). The substances known to modulate BKCa channel activity influenced the bioenergetics of D. discoideum mitochondria. In isolated mitochondria, NS1619 and NS11021 stimulated non-phosphorylating respiration and depolarized membrane potential, indicating the channel activation. These effects were blocked by iberiotoxin and paxilline. Moreover, the activation of the channel resulted in attenuation of superoxide formation, but its inhibition had the opposite effect. Immunological analysis with antibodies raised against mammalian BKCa channel subunits detected a pore-forming α subunit and auxiliary β subunits of the channel in D. discoideum mitochondria. In conclusion, we show for the first time that mitochondria of D. discoideum, a unicellular ameboid protozoon that facultatively forms multicellular structures, contain a large-conductance calcium-activated potassium channel with electrophysiological, biochemical and molecular properties similar to those of the channels previously described in mammalian and plant mitochondria.

  16. 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. Copyright © 2012 Elsevier GmbH. All rights reserved.

  17. Facilitation of ß-cell K(ATP) channel sulfonylurea sensitivity by a cAMP analog selective for the cAMP-regulated guanine nucleotide exchange factor Epac.

    PubMed

    Leech, Colin A; Dzhura, Igor; Chepurny, Oleg G; Schwede, Frank; Genieser, Hans-G; Holz, George G

    2010-01-01

    Clinical studies demonstrate that combined administration of sulfonylureas with exenatide can induce hypoglycemia in type 2 diabetic subjects. Whereas sulfonylureas inhibit ß-cell K(ATP) channels by binding to the sulfonylurea receptor-1 (SUR1), exenatide binds to the GLP-1 receptor, stimulates ß-cell cAMP production and activates both PKA and Epac. In this study, we hypothesized that the adverse in vivo interaction of sulfonylureas and exenatide to produce hypoglycemia might be explained by Epac-mediated facilitation of K(ATP) channel sulfonylurea sensitivity. We now report that the inhibitory action of a sulfonylurea (tolbutamide) at K(ATP) channels was facilitated by 2’-O-Me-cAMP, a selective activator of Epac. Thus, under conditions of excised patch recording, the dose-response relationship describing the inhibitory action of tolbutamide at human ß-cell or rat INS-1 cell K(ATP) channels was left-shifted in the presence of 2’-O-Me-cAMP, and this effect was abolished in INS-1 cells expressing a dominant-negative Epac2. Using an acetoxymethyl ester prodrug of an Epac-selective cAMP analog (8-pCP T-2’-O-Me-cAMP-AM), the synergistic interaction of an Epac activator and tolbutamide to depolarize INS-1 cells and to raise [Ca²(+)](i) was also measured. This effect of 8-pCP T-2’-O-Me-cAMP-AM correlated with its ability to stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis that might contribute to the changes in K(ATP) channel sulfonylurea-sensitivity reported here. On the basis of such findings, we propose that the adverse interaction of sulfonylureas and exenatide to induce hypoglycemia involves at least in part, a functional interaction of these two compounds to close K(ATP) channels, to depolarize ß-cells and to promote insulin secretion.

  18. Optogenetic techniques for the study of native potassium channels.

    PubMed

    Sandoz, Guillaume; Levitz, Joshua

    2013-01-01

    Optogenetic tools were originally designed to target specific neurons for remote control of their activity by light and have largely been built around opsin-based channels and pumps. These naturally photosensitive opsins are microbial in origin and are unable to mimic the properties of native neuronal receptors and channels. Over the last 8 years, photoswitchable tethered ligands (PTLs) have enabled fast and reversible control of mammalian ion channels, allowing optical control of neuronal activity. One such PTL, maleimide-azobenzene-quaternary ammonium (MAQ), contains a maleimide (M) to tether the molecule to a genetically engineered cysteine, a photoisomerizable azobenzene (A) linker and a pore-blocking quaternary ammonium group (Q). MAQ was originally used to photocontrol SPARK, an engineered light-gated potassium channel derived from Shaker. Potassium channel photoblock by MAQ has recently been extended to a diverse set of mammalian potassium channels including channels in the voltage-gated and K2P families. Photoswitchable potassium channels, which maintain native properties, pave the way for the optical control of specific aspects of neuronal function and for high precision probing of a specific channel's physiological functions. To extend optical control to natively expressed channels, without overexpression, one possibility is to develop a knock-in mouse in which the wild-type channel gene is replaced by its light-gated version. Alternatively, the recently developed photoswitchable conditional subunit technique provides photocontrol of the channel of interest by molecular replacement of wild-type complexes. Finally, photochromic ligands also allow photocontrol of potassium channels without genetic manipulation using soluble compounds. In this review we discuss different techniques for optical control of native potassium channels and their associated advantages and disadvantages.

  19. Activation of Adenosine Triphosphate-regulated Potassium Channels during Reperfusion Restores Isoflurane Postconditioning-induced Cardiac Protection in Acutely Hyperglycemic Rabbits.

    PubMed

    Raphael, Jacob; Gozal, Yaacov; Navot, Nachum; Zuo, Zhiyi

    2015-06-01

    Hyperglycemia is known to inhibit myocardial anesthetic postconditioning. The authors tested whether activation of adenosine triphosphate-regulated potassium (KATP) channels would restore anesthetic postconditioning during acute hyperglycemia. Rabbits subjected to 40-min myocardial ischemia and 3-h reperfusion (ischemia-reperfusion [I/R]) were assigned to groups (n = 10 in each group) with or without isoflurane postconditioning (2.1% for 5 min) in the presence or absence of hyperglycemia and/or the KATP channel agonist diazoxide. Creatine kinase MB fraction and infarct size were measured. Phosphorylated protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) were assessed. Oxidative stress was evaluated by measuring malondialdehyde, and apoptosis was assessed by dUTP nick-end labeling and activated caspase-3. Postconditioning significantly reduced myocardial infarct size (26 ± 4% in the isoflurane [ISO] group vs. 53 ± 2% in the I/R group; P = 0.007); whereas, hyperglycemia inhibited this effect (infarct size: 47 ± 2%, P = 0.02 vs. the ISO group). Phosphorylated and eNOS levels increased, whereas malondialdehyde and myocardial apoptosis were significantly lower after isoflurane postconditioning compared with I/R. These effects were inhibited by acute hyperglycemia. Diazoxide restored the protective effect of isoflurane in the hyperglycemic animals (infarct size: 29 ± 2%; P = 0.01 vs. the I/R group), reduced malondialdehyde levels and myocardial apoptosis, but did not affect the expression of phosphorylated Akt or eNOS. KATP channel activation restored anesthetic postconditioning-induced myocardial protection under acute hyperglycemia. This effect occurred without increasing Akt or eNOS phosphorylation, suggesting that KATP channels are located downstream to Akt and eNOS in the pathway of isoflurane-induced myocardial postconditioning.

  20. Local antinociceptive action of fluoxetine in the rat formalin assay: Role of L-arginine-nitric oxide-cGMP-KATP channel pathway.

    PubMed

    Ghorbanzadeh, Behnam; Mansouri, Mohammad Taghi; Naghizadeh, Bahareh; Alboghobeish, Soheila

    2017-08-08

    The present study was conducted to evaluate the local antinociceptive actions of fluoxetine, a selective serotonin reuptake inhibitor, and the possible involvement of the L-arginine/NO/cGMP/KATP channel pathway in this effect using formalin test in rats. To elucidate the underlying mechanisms, animals were pre-treated with L-NAME, aminoguanidine, methylene blue, glibenclamide, L-arginine, sodium nitroprusside, or diazoxide. Local ipsilateral, but not contralateral, administration of fluoxetine (10-300 μg/paw) dose-dependently suppressed flinching number during both early and late phases of the test, and this was comparable to morphine also given peripherally. Pre-treatment with L-NAME, aminoguanidine, methylene blue or glibenclamide dose-dependently prevented fluoxetine (100 μg/paw)-induced antinociception in late phase. On the other hand, administration of L-arginine, sodium nitroprusside and diazoxide significantly enhanced the antinociception caused by fluoxetine in the late phase of the test. However, these treatments had no significant effect on the antinociceptive response of fluoxetine in the early phase of formalin test. Our data demonstrate that local peripheral antinociception of fluoxetine during the late phase of formalin test could be due to activation of L-arginine/NO/cGMP/KATP channel pathway. The peripheral action of fluoxetine raises the possibility that topical application of this drug (e.g. as a cream, ointment or jelly) may be a useful method for relieving the inflammatory pain states.

  1. Chronic nicotine blunts hypoxic sensitivity in perinatal rat adrenal chromaffin cells via upregulation of KATP channels: role of alpha7 nicotinic acetylcholine receptor and hypoxia-inducible factor-2alpha.

    PubMed

    Buttigieg, Josef; Brown, Stephen; Holloway, Alison C; Nurse, Colin A

    2009-06-03

    Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of K(ATP) channels via stimulation of alpha7 nicotinic ACh receptors (AChRs). These K(ATP) channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K(+) current, membrane depolarization, rise in cytosolic Ca(2+), and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of K(ATP) channels, was present. Both K(ATP) current density and K(ATP) channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for approximately 1 week, and was prevented by coincubation with selective blockers of alpha7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (>90%) of the transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha), suggesting involvement of the HIF pathway. The therapeutic potential of K(ATP) channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

  2. Optogenetic techniques for the study of native potassium channels

    PubMed Central

    Sandoz, Guillaume; Levitz, Joshua

    2013-01-01

    Optogenetic tools were originally designed to target specific neurons for remote control of their activity by light and have largely been built around opsin-based channels and pumps. These naturally photosensitive opsins are microbial in origin and are unable to mimic the properties of native neuronal receptors and channels. Over the last 8 years, photoswitchable tethered ligands (PTLs) have enabled fast and reversible control of mammalian ion channels, allowing optical control of neuronal activity. One such PTL, maleimide-azobenzene-quaternary ammonium (MAQ), contains a maleimide (M) to tether the molecule to a genetically engineered cysteine, a photoisomerizable azobenzene (A) linker and a pore-blocking quaternary ammonium group (Q). MAQ was originally used to photocontrol SPARK, an engineered light-gated potassium channel derived from Shaker. Potassium channel photoblock by MAQ has recently been extended to a diverse set of mammalian potassium channels including channels in the voltage-gated and K2P families. Photoswitchable potassium channels, which maintain native properties, pave the way for the optical control of specific aspects of neuronal function and for high precision probing of a specific channel’s physiological functions. To extend optical control to natively expressed channels, without overexpression, one possibility is to develop a knock-in mouse in which the wild-type channel gene is replaced by its light-gated version. Alternatively, the recently developed photoswitchable conditional subunit technique provides photocontrol of the channel of interest by molecular replacement of wild-type complexes. Finally, photochromic ligands also allow photocontrol of potassium channels without genetic manipulation using soluble compounds. In this review we discuss different techniques for optical control of native potassium channels and their associated advantages and disadvantages. PMID:23596388

  3. Endoplasmic reticulum potassium-hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes.

    PubMed

    Kuum, Malle; Veksler, Vladimir; Liiv, Joanna; Ventura-Clapier, Renee; Kaasik, Allen

    2012-02-01

    Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation-proton exchangers could act as routes for such a re-entry. ER Ca(2+) uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K(+) in the uptake solution with Na(+) or tetraethylammonium led to a strong inhibition of Ca(2+) uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium-proton exchanger (quinine or propranolol) but not of the sodium-proton exchanger reduced ER Ca(2+) uptake by 56-82%. Externally added nigericin, a potassium-proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K(+) (SK(Ca)) channels (UCL 1684, dequalinium) blocked the uptake of Ca(2+) by the ER in all preparations by 48-94%, whereas inhibitors of other K(+) channels (IK(Ca), BK(Ca) and K(ATP)) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SK(Ca) channels and the potassium-proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SK(Ca) channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca(2+) uptake.

  4. Role of renal vascular potassium channels in physiology and pathophysiology.

    PubMed

    Salomonsson, Max; Brasen, Jens Christian; Sorensen, Charlotte M

    2017-03-30

    The control of renal vascular tone is important for the regulation of salt and water balance, blood pressure and the protection against damaging elevated glomerular pressure. The K(+) conductance is a major factor in the regulation of the membrane potential (Vm ) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by Vm via its effect on the opening probability of voltage operated Ca(2+) channels (VOCC) in VSMC. When K(+) conductance increases Vm becomes more negative and vasodilation follows, while deactivation of K(+) channels leads to depolarization and vasoconstriction. K(+) channels in EC indirectly participate in the control of vascular tone by endothelium derived vasodilation. Therefore, by regulating the tone of renal resistance vessels, K(+) channels have a potential role in the control of fluid homeostasis and blood pressure as well as in the protection of the renal parenchyma. The main classes of K(+) channels (calcium activated (KCa ), inward rectifier (Kir ), voltage activated (Kv ) and ATP sensitive (KATP )) have been found in the renal vessels. In this review, we summarize results available in the literature and our own studies in the field. We compare the ambiguous in vitro and in vivo results. We discuss the role of single types of K(+) channels and the integrated function of several classes. We also deal with the possible role of renal vascular K(+) channels in the pathophysiology of hypertension, diabetes mellitus and sepsis. This article is protected by copyright. All rights reserved.

  5. The novel H2S-donor 4-carboxyphenyl isothiocyanate promotes cardioprotective effects against ischemia/reperfusion injury through activation of mitoKATP channels and reduction of oxidative stress.

    PubMed

    Testai, Lara; Marino, Alice; Piano, Ilaria; Brancaleone, Vincenzo; Tomita, Kengo; Di Cesare Mannelli, Lorenzo; Martelli, Alma; Citi, Valentina; Breschi, Maria C; Levi, Roberto; Gargini, Claudia; Bucci, Mariarosaria; Cirino, Giuseppe; Ghelardini, Carla; Calderone, Vincenzo

    2016-11-01

    The endogenous gasotransmitter hydrogen sulphide (H2S) is an important regulator of the cardiovascular system, particularly of myocardial function. Moreover, H2S exhibits cardioprotective activity against ischemia/reperfusion (I/R) or hypoxic injury, and is considered an important mediator of "ischemic preconditioning", through activation of mitochondrial potassium channels, reduction of oxidative stress, activation of the endogenous "anti-oxidant machinery" and limitation of inflammatory responses. Accordingly, H2S-donors, i.e. pro-drugs able to generate exogenous H2S, are viewed as promising therapeutic agents for a number of cardiovascular diseases. The novel H2S-donor 4-carboxy phenyl-isothiocyanate (4CPI), whose vasorelaxing effects were recently reported, was tested here in different experimental models of myocardial I/R. In Langendorff-perfused rat hearts subjected to I/R, 4CPI significantly improved the post-ischemic recovery of myocardial functional parameters and limited tissue injury. These effects were antagonized by 5-hydroxydecanoic acid (a blocker of mitoKATP channels). Moreover, 4CPI inhibited the formation of reactive oxygen species. We found the whole battery of H2S-producing enzymes to be present in myocardial tissue: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). Notably, 4CPI down-regulated the post-ischemic expression of CSE. In Langendorff-perfused mouse hearts, 4CPI reduced the post-ischemic release of norepinephrine and the incidence of ventricular arrhythmias. In both rat and mouse hearts, 4CPI did not affect the degranulation of resident mast cells. In isolated rat cardiac mitochondria, 4CPI partially depolarized the mitochondrial membrane potential; this effect was antagonized by ATP (i.e., the physiological inhibitor of KATP channels). Moreover, 4CPI abrogated calcium uptake in the mitochondrial matrix. Finally, in an in vivo model of acute myocardial infarction in rats, 4

  6. Lidocaine relaxation in isolated rat aortic rings is enhanced by endothelial removal: possible role of Kv, KATP channels and A2a receptor crosstalk.

    PubMed

    Arsyad, Aryadi; Dobson, Geoffrey P

    2016-12-03

    Lidocaine is an approved local anesthetic and Class 1B antiarrhythmic with a number of ancillary properties. Our aim was to investigate lidocaine's vasoreactivity properties in intact versus denuded rat thoracic aortic rings, and the effect of inhibitors of nitric oxide (NO), prostenoids, voltage-dependent Kv and KATP channels, membrane Na(+)/K(+) pump, and A2a and A2b receptors. Aortic rings were harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, pH 7.4, 37 °C. The rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing lidocaine concentrations was examined. Rings were tested for viability after each experiment with maximally dilating 100 μM papaverine. The drugs 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined. All drugs tested had no significant effect on basal tension. Lidocaine relaxation in intact rings was biphasic between 1 and 10 μM (Phase 1) and 10 and 1000 μM (Phase 2). Mechanical removal of the endothelium resulted in further relaxation, and at lower concentrations ring sensitivity (% relaxation per μM lidocaine) significantly increased 3.5 times compared to intact rings. The relaxing factor(s) responsible for enhancing ring relaxation did not appear to be NO- or prostacyclin-dependent, as L-NAME and indomethacin had little or no effect on intact ring relaxation. In denuded rings, lidocaine relaxation was completely abolished by Kv channel inhibition and significantly reduced by antagonists of the MitoKATP channel, and to a lesser extent the SarcKATP channel. Curiously, A2a subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 μM, but not the A2b receptor. We show that lidocaine relaxation in rat thoracic aorta was biphasic and significantly enhanced by endothelial removal, which did not appear to be NO or

  7. No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation.

    PubMed

    Brunt, Vienna E; Fujii, Naoto; Minson, Christopher T

    2013-11-01

    In human cutaneous microvasculature, endothelium-derived hyperpolarizing factors (EDHFs) account for a large portion of vasodilation associated with local stimuli. Thus we sought to determine the role of EDHFs in active vasodilation (AVD) to passive heating in two protocols. Whole body heating was achieved using water-perfused suits (core temperature increase of 0.8-1.0°C), and skin blood flow was measured using laser-Doppler flowmetry. In the first protocol, four sites were perfused continuously via microdialysis with: 1) control; 2) tetraethylammonium (TEA) to block calcium-activated potassium (KCa) channels, and thus the actions of EDHFs; 3) N-nitro-l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase (NOS); and 4) TEA + l-NAME (n = 8). Data are presented as percent maximal cutaneous vascular conductance (CVC). TEA had no effect on AVD (CVC during heated plateau: control 57.4 ± 4.9% vs. TEA 63.2 ± 5.2%, P = 0.27), indicating EDHFs are not obligatory. l-NAME attenuated plateau CVC to 33.7 ± 5.4% (P < 0.01 vs. control); while TEA + l-NAME augmented plateau CVC compared with l-NAME alone (49.7 ± 5.3%, P = 0.02). From these data, it appears combined blockade of EDHFs and NOS necessitates dilation through other means, possibly through inward rectifier (KIR) and/or ATP-sensitive (KATP) potassium channels. To test this second hypothesis, we measured AVD at the following sites (n = 8): 1) control, 2) l-NAME, 3) l-NAME + TEA, and 4) l-NAME + TEA + barium chloride (BaCl2; KIR and KATP blocker). The addition of BaCl2 to l-NAME + TEA reduced plateau CVC to 32.7 ± 6.6% (P = 0.02 vs. l-NAME + TEA), which did not differ from the l-NAME site. These data combined demonstrate a complex interplay between vasodilatory pathways, with cross-talk between NO, KCa channels, and KIR and/or KATP channels.

  8. No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation

    PubMed Central

    Brunt, Vienna E.; Fujii, Naoto

    2013-01-01

    In human cutaneous microvasculature, endothelium-derived hyperpolarizing factors (EDHFs) account for a large portion of vasodilation associated with local stimuli. Thus we sought to determine the role of EDHFs in active vasodilation (AVD) to passive heating in two protocols. Whole body heating was achieved using water-perfused suits (core temperature increase of 0.8–1.0°C), and skin blood flow was measured using laser-Doppler flowmetry. In the first protocol, four sites were perfused continuously via microdialysis with: 1) control; 2) tetraethylammonium (TEA) to block calcium-activated potassium (KCa) channels, and thus the actions of EDHFs; 3) N-nitro-l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase (NOS); and 4) TEA + l-NAME (n = 8). Data are presented as percent maximal cutaneous vascular conductance (CVC). TEA had no effect on AVD (CVC during heated plateau: control 57.4 ± 4.9% vs. TEA 63.2 ± 5.2%, P = 0.27), indicating EDHFs are not obligatory. l-NAME attenuated plateau CVC to 33.7 ± 5.4% (P < 0.01 vs. control); while TEA + l-NAME augmented plateau CVC compared with l-NAME alone (49.7 ± 5.3%, P = 0.02). From these data, it appears combined blockade of EDHFs and NOS necessitates dilation through other means, possibly through inward rectifier (KIR) and/or ATP-sensitive (KATP) potassium channels. To test this second hypothesis, we measured AVD at the following sites (n = 8): 1) control, 2) l-NAME, 3) l-NAME + TEA, and 4) l-NAME + TEA + barium chloride (BaCl2; KIR and KATP blocker). The addition of BaCl2 to l-NAME + TEA reduced plateau CVC to 32.7 ± 6.6% (P = 0.02 vs. l-NAME + TEA), which did not differ from the l-NAME site. These data combined demonstrate a complex interplay between vasodilatory pathways, with cross-talk between NO, KCa channels, and KIR and/or KATP channels. PMID:23970531

  9. 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.

  10. Potassium channel in the mitochondria of human keratinocytes.

    PubMed

    Toczyłowska-Mamińska, Renata; Olszewska, Anna; Laskowski, Michał; Bednarczyk, Piotr; Skowronek, Krzysztof; Szewczyk, Adam

    2014-03-01

    The activation of mitochondrial potassium channels induces cytoprotection in various cell types. Hence, the identification of ion channels present in the inner mitochondrial membrane of keratinocytes is important in distinguishing possible protective mechanisms in these cells. In this paper, inner membrane mitochondrial ion channels of the human keratinocyte HaCaT cell line were investigated using a patch-clamp technique. We observed potassium-selective channel activity with a conductance of 83 pS at positive voltages. The I-V curve indicates that the observed channel has rectifying properties. Moreover, the channel activity was inhibited by acidic pH and 1 mM lidocaine. Using reverse transcriptase-PCR, we found an mRNA transcript for the TASK-3 (tandem pore domain acid-sensitive K channels) channel. We observed co-localization of the TASK-3 protein and a mitochondrial marker in the mitochondria of HaCaT cells. Additionally, we showed that TASK-3 knockdown HaCaT cells markedly decreased viability after UVB radiation exposure compared with control cells. In summary, the single-channel activity and properties of a mitochondrial potassium channel in a keratinocyte HaCaT cell line have been described.

  11. Targeting BK (big potassium) Channels in Epilepsy

    PubMed Central

    N'Gouemo, Prosper

    2011-01-01

    Introduction Epilepsies are disorders of neuronal excitability characterized by spontaneous and recurrent seizures. Ion channels are critical for regulating neuronal excitability and, therefore, can contribute significantly to epilepsy pathophysiology. In particular, large conductance, Ca2+-activated K+ (BKCa) channels play an important role in seizure etiology. These channels are activated by both membrane depolarization and increased intracellular Ca2+. This unique coupling of Ca2+ signaling to membrane depolarization is important in controlling neuronal hyperexcitability, as outward K+ current through BKCa channels hyperpolarizes neurons. Areas covered This review focuses on BKCa channel structure-function and discusses the role of these channels in epilepsy pathophysiology. Expert opinion Loss-of-function BKCa channels contribute neuronal hyperexcitability that can lead to temporal lobe epilepsy, tonic-clonic seizures and alcohol withdrawal seizures. Similarly, BKCa channel blockade can trigger seizures and status epilepticus. Paradoxically, some mutations in BKCa channel subunit can give rise to the channel gain-of-function that leads to development of idiopathic epilepsy (primarily absence epilepsy). Seizures themselves also enhance BKCa channel currents associated with neuronal hyperexcitability, and blocking BKCa channels suppresses generalized tonic-clonic seizures. Thus, both loss-of-function and gain-of-function BKCa channels might serve as molecular targets for drugs to suppress certain seizure phenotypes including temporal lobe seizures and absence seizures, respectively. PMID:21923633

  12. Functional diversity of potassium channel voltage-sensing domains

    PubMed Central

    Islas, León D.

    2016-01-01

    Abstract Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology. PMID:26794852

  13. Functional diversity of potassium channel voltage-sensing domains.

    PubMed

    Islas, León D

    2016-01-01

    Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.

  14. 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.

  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.

    PubMed

    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

    2011-09-01

    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 K(ATP) 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 K(ATP) channels in the CNS. Apparently, circulating insulin stimulates fatty acid uptake in WAT but not in BAT, directly and indirectly through the CNS.

  16. 17β-Estradiol Rapidly Increases KATP Activity in GnRH via a Protein Kinase Signaling Pathway

    PubMed Central

    Zhang, Chunguang; Kelly, Martin J.; Rønnekleiv, Oline K.

    2010-01-01

    17β-Estradiol (E2) both inhibits and excites GnRH neurons via presynaptic as well as postsynaptic mechanisms. Although it has been demonstrated that E2 can alter the excitability of GnRH neurons via direct actions, the intracellular signaling cascades mediating these actions are not well understood. Previously we have shown that the activity of one of the critical ion channels needed for maintaining GnRH neurons in a hyperpolarized state, the ATP-sensitive potassium channel (KATP) channel, is augmented by E2 in ovariectomized females. However, the mRNA expression of the KATP channel subunits Kir6.2 and SUR1 are unchanged with in vivo E2 treatment. Therefore, to elucidate the cellular signaling mechanism(s) modulating the channel activity, we did whole-cell patch-clamp recording of enhanced green fluorescent protein-GnRH neurons from ovariectomized female mice to study the acute effects of E2. E2 dose-dependently (EC50 = 0.6 nM) enhanced the diazoxide (channel opener)-activated KATP channel currents by 1.2- to 2.0-fold, which was antagonized by ICI 182,780. E2-BSA was equally as effective as E2, whereas E2 had no effect. The protein kinase A (PKA) activator forskolin mimicked the effects of E2, whereas the PKA inhibitor H89 and the protein kinase C (PKC) inhibitor bisindolylmaleimide I blocked the effects of E2. Similar to E2, STX, a membrane estrogen receptor (ER) agonist that does not bind to ERα or ERβ, also potentiated the diazoxide-induced KATP channel current by 1.5-fold. Therefore, E2 can potentiate KATP channel activity in GnRH neurons through a membrane ER-activated PKC-PKA signaling pathway. PMID:20660067

  17. Pore size matters for potassium channel conductance

    PubMed Central

    Moldenhauer, Hans; Pincuntureo, Matías

    2016-01-01

    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. PMID:27619418

  18. 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.

  19. 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

  20. Active relaxation of human gallbladder muscle is mediated by ATP-sensitive potassium channels.

    PubMed

    Bird, N C; Ahmed, R; Chess-Williams, R; Johnson, A G

    2002-01-01

    Active and significant relaxation of the human gallbladder must be one of the facets of its motility during both the filling and emptying cycle. Conflicting reports about the presence or significance of nitric oxide have been reported in the literature. The aim of this study was to investigate the role of nitric oxide and K(ATP) channels in human gallbladder muscle using isolated strips from human gallbladder. Full thickness strips were obtained from 56 human gallbladders and suspended under isometric tension in organ baths. The effect of nitric oxide donors and inhibitors on cholecystokinin octapeptide- and carbachol-induced contraction was examined. In separate experiments the effect of the K(ATP) channel activator, cromakalim, and the inhibitor, glibenclamide, were determined. Cromakalim induced a significant relaxation of agonist-induced contraction in human gallbladder in vitro, an effect which was abolished by the K(ATP) channel inhibitor glibenclamide. No evidence of significant nitric oxide involvement in relaxation was observed. This study has demonstrated the presence of K(ATP) channels in human gallbladder for the first time. These are capable of causing significant relaxation in the presence of hormonal and muscarinic agonists and may represent a major pathway for gallbladder relaxation. Copyright 2002 S. Karger AG, Basel

  1. 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

  2. Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus

    PubMed Central

    Zawar, C; Plant, T D; Schirra, C; Konnerth, A; Neumcke, B

    1999-01-01

    The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling.The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0·3 mm), a KATP channel opener, and to tolbutamide (0·5 mm) or glibenclamide (20 μm), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89% of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution.Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channelsmay be formed in 75%of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones).The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied. PMID:9852317

  3. Ion conduction pore is conserved among potassium channels

    NASA Astrophysics Data System (ADS)

    Lu, Zhe; Klem, Angela M.; Ramu, Yajamana

    2001-10-01

    Potassium channels, a group of specialized membrane proteins, enable K+ ions to flow selectively across cell membranes. Transmembrane K+ currents underlie electrical signalling in neurons and other excitable cells. The atomic structure of a bacterial K+ channel pore has been solved by means of X-ray crystallography. To the extent that the prokaryotic pore is representative of other K+ channels, this landmark achievement has profound implications for our general understanding of K+ channels. But serious doubts have been raised concerning whether the prokaryotic K+ channel pore does actually represent those of eukaryotes. Here we have addressed this fundamental issue by substituting the prokaryotic pore into eukaryotic voltage-gated and inward-rectifier K+ channels. The resulting chimaeras retain the respective functional hallmarks of the eukaryotic channels, which indicates that the ion conduction pore is indeed conserved among K+ channels.

  4. Loss-of-Function Mutations in the KCNJ8-Encoded Kir6.1 KATP Channel and Sudden Infant Death Syndrome

    PubMed Central

    Tester, David J.; Tan, Bi-Hua; Medeiros-Domingo, Argelia; Song, Chunhua; Makielski, Jonathan C.; Ackerman, Michael J.

    2011-01-01

    Background Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8-encoded Kir6.1 (KATP) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8-deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. Methods and Results Using PCR, DHPLC, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white, age 2.9 ± 1.9 months). KCNJ8 mutations were co-expressed heterologously with SUR2A in COS-1 cells and characterized using whole cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues, and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared to WT, the pinacidil-activated KATP current was decreased 45% to 68% for Kir6.1-E322del and 40% to 57% for V346I between -20 mV to 40 mV. Conclusions Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency. PMID:21836131

  5. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Inhibition of Kir4.1 potassium channels by quinacrine.

    PubMed

    Marmolejo-Murillo, Leticia G; Aréchiga-Figueroa, Iván A; Cui, Meng; Moreno-Galindo, Eloy G; Navarro-Polanco, Ricardo A; Sánchez-Chapula, José A; Ferrer, Tania; Rodríguez-Menchaca, Aldo A

    2017-05-15

    Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3μM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.

  7. The antipsychotic drug chlorpromazine inhibits HERG potassium channels

    PubMed Central

    Thomas, Dierk; Wu, Kezhong; Kathöfer, Sven; Katus, Hugo A; Schoels, Wolfgang; Kiehn, Johann; Karle, Christoph A

    2003-01-01

    Acquired long QT syndrome (aLQTS) is caused by prolongation of the cardiac action potential because of blockade of cardiac ion channels and delayed repolarization of the heart. Patients with aLQTS carry an increased risk for torsade de pointes arrhythmias and sudden cardiac death. Several antipsychotic drugs may cause aLQTS. Recently, cases of QTc prolongation and torsade de pointes associated with chlorpromazine treatment have been reported. Blockade of human ether-a-go-go-related gene (HERG) potassium channels, which plays a central role in arrhythmogenesis, has previously been reported to occur with chlorpromazine, but information on the mechanism of block is currently not available. We investigated the effects of chlorpromazine on cloned HERG potassium channels to determine the biophysical mechanism of block. HERG channels were heterologously expressed in Xenopus laevis oocytes, and ion currents were measured using the two-microelectrode voltage-clamp technique. Chlorpromazine blocked HERG potassium channels with an IC50 value of 21.6 μM and a Hill coefficient of 1.11. Analysis of the voltage dependence of block revealed a reduction of inhibition at positive membrane potentials. Inhibition of HERG channels by chlorpromazine displayed reverse frequency dependence, that is, the amount of block was lower at higher stimulation rates. No marked changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or changes of the inactivation time constant were observed. In conclusion, HERG channels were blocked in the closed and activated states, and unblocking occurred very slowly. PMID:12788816

  8. 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.

  9. Interaction of mitochondrial potassium channels with the permeability transition pore.

    PubMed

    Cheng, Yu; Debska-Vielhaber, Grazyna; Siemen, Detlef

    2010-05-17

    Three types of potassium channels cooperate with the permeability transition pore (PTP) in the inner mitochondrial membranes of various tissues, mtK((ATP)), mtBK, and mtKv1.3. While the latter two share similarities with their plasma membrane counterparts, mtK((ATP)) exhibits considerable differences with the plasma membrane K((ATP))-channel. One important function seems to be suppression of release of proapototic substances from mitochondria through the PTP. Open potassium channels tend to keep the PTP closed thus acting as antiapoptotic. Nevertheless, in their mode of action there are considerable differences among them. This review introduces three K(+)-channels and the PTP, and discusses known facts about their interaction. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Opening of Astrocytic Mitochondrial ATP-Sensitive Potassium Channels Upregulates Electrical Coupling between Hippocampal Astrocytes in Rat Brain Slices

    PubMed Central

    Wang, Jiangping; Li, Zhongxia; Feng, Mei; Ren, Keming; Shen, Guoxia; Zhao, Congying; Jin, Xiaoming; Jiang, Kewen

    2013-01-01

    Astrocytes form extensive intercellular networks through gap junctions to support both biochemical and electrical coupling between adjacent cells. ATP-sensitive K+ (KATP) channels couple cell metabolic state to membrane excitability and are enriched in glial cells. Activation of astrocytic mitochondrial KATP (mitoKATP) channel regulates certain astrocytic functions. However, less is known about its impact on electrical coupling between directly coupled astrocytes ex vivo. By using dual patch clamp recording, we found that activation of mitoKATP channel increased the electrical coupling ratio in brain slices. The electrical coupling ratio started to increase 3 min after exposure to Diazoxide, a mitoKATP channel activator, peaked at 5 min, and maintained its level with little adaptation until the end of the 10-min treatment. Blocking the mitoKATP channel with 5-hydroxydecanoate, inhibited electrical coupling immediately, and by 10-min, the ratio dropped by 71% of the initial level. Activation of mitoKATP channel also decreased the latency time of the transjunctional currents by 50%. The increase in the coupling ratio resulting from the activation of the mitoKATP channel in a single astrocyte was further potentiated by the concurrent inhibiting of the channel on the recipient astrocyte. Furthermore, Meclofenamic acid, a gap-junction inhibitor which completely blocked the tracer coupling, hardly reversed the impact of mitoKATP channel's activation on electrical coupling (by 7%). The level of mitochondrial Connexin43, a gap junctional subunit, significantly increased by 70% in astrocytes after 10-min Diazoxide treatment. Phospho-ERK signals were detected in Connexin43 immunoprecipitates in the Diazoxide-treated astrocytes, but not untreated control samples. Finally, inhibiting ERK could attenuate the effects of Diazoxide on electrical coupling by 61%. These findings demonstrate that activation of astrocytic mitoKATP channel upregulates electrical coupling between

  11. Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

    PubMed Central

    Félétou, Michel

    2009-01-01

    The three subtypes of calcium-activated potassium channels (KCa) of large, intermediate and small conductance (BKCa, IKCa and SKCa) are present in the vascular wall. In healthy arteries, BKCa channels are preferentially expressed in vascular smooth muscle cells, while IKCa and SKCa are preferentially located in endothelial cells. The activation of endothelial IKCa and SKCa contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na+/K+-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H2O2) hyperpolarize and relax the underlying smooth muscle cells by activating BKCa. In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BKCa. Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle KCa could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IKCa may prevent restenosis and that of BKCa channels sepsis-dependent hypotension. PMID:19187341

  12. Exchange protein activated by cAMP (Epac) mediates cAMP-dependent but protein kinase A-insensitive modulation of vascular ATP-sensitive potassium channels.

    PubMed

    Purves, Gregor I; Kamishima, Tomoko; Davies, Lowri M; Quayle, John M; Dart, Caroline

    2009-07-15

    Exchange proteins directly activated by cyclic AMP (Epacs or cAMP-GEF) represent a family of novel cAMP-binding effector proteins. The identification of Epacs and the recent development of pharmacological tools that discriminate between cAMP-mediated pathways have revealed previously unrecognized roles for cAMP that are independent of its traditional target cAMP-dependent protein kinase (PKA). Here we show that Epac exists in a complex with vascular ATP-sensitive potassium (KATP) channel subunits and that cAMP-mediated activation of Epac modulates KATP channel activity via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive protein phosphatase 2B (PP-2B, calcineurin). Application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP, at concentrations that activate Epac but not PKA, caused a 41.6 +/- 4.7% inhibition (mean +/- S.E.M.; n = 7) of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Importantly, similar results were obtained when cAMP was elevated by addition of the adenylyl cyclase activator forskolin in the presence of the structurally distinct PKA inhibitors, Rp-cAMPS or KT5720. Activation of Epac by 8-pCPT-2'-O-Me-cAMP caused a transient 171.0 +/- 18.0 nM (n = 5) increase in intracellular Ca2+ in Fura-2-loaded aortic myocytes, which persisted in the absence of extracellular Ca2+. Inclusion of the Ca2+-specific chelator BAPTA in the pipette-filling solution or preincubation with the calcineurin inhibitors, cyclosporin A or ascomycin, significantly reduced the ability of 8-pCPT-2'-O-Me-cAMP to inhibit whole-cell KATP currents. These results highlight a previously undescribed cAMP-dependent regulatory mechanism that may be essential for understanding the physiological and pathophysiological roles ascribed to arterial KATP channels in the control of vascular tone and blood flow.

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

    PubMed

    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.

  14. 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

  15. Toxicology of potassium channel-directed compounds in mosquitoes.

    PubMed

    Larson, Nicholas R; Carlier, Paul R; Gross, Aaron D; Islam, Rafique M; Ma, Ming; Sun, Baonan; Totrov, Maxim M; Yadav, Roopali; Bloomquist, Jeffrey R

    2016-06-01

    Potential targets for new vector control insecticides are nerve and muscle potassium channels. In this study, the activities of known potassium channel blockers (4-aminopyridine, quinidine, and tetraethylammonium) and the insecticide propoxur were compared to three experimental catechols and several other compounds against Anopheles gambiae and Aedes aegypti mosquitoes. Experimental catechol 1 was the most toxic experimental compound in all of the mortality assays conducted, but was at least 100-fold and 39-fold less toxic than propoxur against Ae. aegypti and An. gambiae, respectively. Injection treatment and synergist (piperonyl butoxide) bioassays found that catechol toxicity was not unduly impacted by cuticular transport or oxidative metabolism. Electrophysiological studies showed a decrease in amplitude of evoked muscle contractions, along with an increase in twitch duration at concentrations that increased basal muscle tension (mM). High concentration effects on basal muscle tension were matched by complete depolarization of the muscle membrane potential. Effects on muscle physiology and blockage of Kv2.1 potassium channels in patch clamp experiments were generally consistent with in vivo toxicity, except for 4-aminopyridine, which suggest the involvement of other potassium channel subtypes. Extensive melanization of Anopheles larvae, but not Aedes larvae, occurred from exposure to catechol compounds. Interaction with the phenol oxidase system within insects may be the cause of this melanization, but any contribution to toxicity requires further investigation.

  16. Potassium Channel Complex Autoimmunity Induced by Inhaled Brain Tissue Aerosol

    PubMed Central

    Meeusen, Jeffrey W.; Klein, Christopher J.; Pirko, Istvan; Haselkorn, Keegan E.; Kryzer, Thomas J.; Pittock, Sean J.; Lachance, Daniel H.; Dyck, P. James; Lennon, Vanda A.

    2011-01-01

    Objective Test the hypothesis that autoimmunity induced by inhalation of aerosolized brain tissue caused outbreaks of sensory-predominant polyradiculoneuropathy among swine abattoir employees in Midwestern USA Methods Mice were exposed intranasally, 5 days weekly, to liquefied brain tissue. Serum from exposed mice, patients and unaffected abattoir employees were analyzed for clinically pertinent neural autoantibodies. Results Patients, coworkers and mice exposed to liquefied brain tissue had an autoantibody profile dominated by neural cation channel IgGs. The most compelling link between patients and exposed mice was MRI evidence of grossly swollen spinal nerve roots. Autoantibody responses in patients and mice were dose-dependent and declined after antigen exposure ceased. Autoantibodies detected most frequently, and at high levels, bound to detergent-solubilized macromolecular complexes containing neuronal voltage-gated potassium channels ligated with a high affinity Kv1 channel antagonist, 125I-α-dendrotoxin. Exposed mice exhibited a behavioral phenotype consistent with potassium channel dysfunction recognized in drosophila with mutant (“shaker”) channels: reduced sensitivity to isoflurane-induced anesthesia. Pathological and electrophysiological findings in patients supported peripheral nerve hyperexcitability over destructive axonal loss. The pain-predominant symptoms were consistent with sensory nerve hyperexcitability Interpretation Our observations establish that inhaled neural antigens readily induce neurological autoimmunity and identify voltage-gated potassium channel complexes as a major immunogen. PMID:22451206

  17. 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.

  18. 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

  19. Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria.

    PubMed

    Checchetto, Vanessa; Segalla, Anna; Allorent, Guillaume; La Rocca, Nicoletta; Leanza, Luigi; Giacometti, Giorgio Mario; Uozumi, Nobuyuki; Finazzi, Giovanni; Bergantino, Elisabetta; Szabò, Ildikò

    2012-07-03

    A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.

  20. Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria

    PubMed Central

    Checchetto, Vanessa; Segalla, Anna; Allorent, Guillaume; La Rocca, Nicoletta; Leanza, Luigi; Giacometti, Giorgio Mario; Uozumi, Nobuyuki; Finazzi, Giovanni; Bergantino, Elisabetta; Szabò, Ildikò

    2012-01-01

    A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b6f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis. PMID:22711813

  1. 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

  2. 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

  3. Human K(ATP) channelopathies: diseases of metabolic homeostasis.

    PubMed

    Olson, Timothy M; Terzic, Andre

    2010-07-01

    Assembly of an inward rectifier K+ channel pore (Kir6.1/Kir6.2) and an adenosine triphosphate (ATP)-binding regulatory subunit (SUR1/SUR2A/SUR2B) forms ATP-sensitive K+ (KATP) channel heteromultimers, widely distributed in metabolically active tissues throughout the body. KATP channels are metabolism-gated biosensors functioning as molecular rheostats that adjust membrane potential-dependent functions to match cellular energetic demands. Vital in the adaptive response to (patho)physiological stress, KATP channels serve a homeostatic role ranging from glucose regulation to cardioprotection. Accordingly, genetic variation in KATP channel subunits has been linked to the etiology of life-threatening human diseases. In particular, pathogenic mutations in KATP channels have been identified in insulin secretion disorders, namely, congenital hyperinsulinism and neonatal diabetes. Moreover, KATP channel defects underlie the triad of developmental delay, epilepsy, and neonatal diabetes (DEND syndrome). KATP channelopathies implicated in patients with mechanical and/or electrical heart disease include dilated cardiomyopathy (with ventricular arrhythmia; CMD1O) and adrenergic atrial fibrillation. A common Kir6.2 E23K polymorphism has been associated with late-onset diabetes and as a risk factor for maladaptive cardiac remodeling in the community-at-large and abnormal cardiopulmonary exercise stress performance in patients with heart failure. The overall mutation frequency within KATP channel genes and the spectrum of genotype-phenotype relationships remain to be established, while predicting consequences of a deficit in channel function is becoming increasingly feasible through systems biology approaches. Thus, advances in molecular medicine in the emerging field of human KATP channelopathies offer new opportunities for targeted individualized screening, early diagnosis, and tailored therapy.

  4. [The influence of activation of the ATP-sensitive potassium channels by flocalin on the function of the cardiovascular system].

    PubMed

    Strutyns'kyĭ, R B; Rovenets', R A; Strutyns'ka, N A; Neshcheret, O P; Moĭbenko, O O

    2013-01-01

    In experiments on the anaesthetized dogs the influence of a new fluorine-containing opener of ATP-sensitive potassium (K(ATP)) channels flocalin on the cardiohemodynamic of great animals in vivo was studied. Flocalin introduced intravenously in doses 0.01 - 1.5 mgs/kg. It is shown that it reduces in dose-dependent manner a system arterial pressure, perfusion pressure in coronary artery and general peripheral resistance of vessels with maximal effects on 56.8 +/- 2.7, 22.4 +/- 4.7 and 47.2% +/- 6.5% accordingly at most dose 1.5 mgs/kg. Flocalin causes development of cardiodepressive reactions in heart, that is exhibited in dose-dependent the decrease of pressure in the left ventricle, speed of growth (dP/dt(max)) and reduction (dP/dt(min)) in it's of pressure with maximal effects on 37.1 +/- 5.1, 51.2 +/- 9.4 and 55.6% +/- 6.9% accordingly at introduction of most dose of flocalin. Diminish of the cardiac out put and heart rate with a maximal effects on 23.1% +/-12.7% and 19.2% +/- 1.7% accordingly at a dose 1.0 mgs/kg was shown. It should be noted that considerable reduction of heart rate and general peripheral resistance of vessels takes place only at the large doses of flocalin - 1 and 1.5 mgs/kg. Thus, it is shown that activation of K(ATP) channels by flocalin causes the dose-dependent decrease of pressure in the system of circulation of blood and contraction activity of myocardium.

  5. 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.

  6. 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.

  7. Lack of effect of potassium channel openers on ATP-modulated potassium channels recorded from rat ventromedial hypothalamic neurones.

    PubMed Central

    Sellers, A. J.; Boden, P. R.; Ashford, M. L.

    1992-01-01

    1. Single neuronal cells were freshly isolated from the ventromedial hypothalamic nuclei (VMHN) of the rat brain. Currents through ATP-modulated and large conductance (160 and 250 pS) calcium-activated potassium channels were recorded by the cell-attached and excised inside-out patch techniques. 2. BRL38227 (lemakalim; 30-90 microM) applied to the superfusing medium produced no change in firing rate of isolated glucose-receptive VMHN neurones in cell-attached recordings. 3. BRL38227, at concentrations of between 30-100 microM applied to the intracellular (cytoplasmic) aspect of inside-out patches, had no effect on the activity of ATP-sensitive K+ channels in the absence of ATP or in the presence of a sub-maximal inhibitory concentration (3 mM) of ATP. Cromakalim, pinacidil, minoxidil sulphate and diazoxide also produced no effect under these conditions. 4. The potassium channel openers (KCO's) were tested on ATP-activated potassium channels recorded from a further subpopulation of VMHN neurones. Application of BRL38227 (up to and including 100 microM) to this channel in inside-out patches either in the absence of ATP or when activated by 5 mM ATP had no effect on channel activity. Identical results were obtained with cromakalim and pinacidil. 5. BRL38227 had no effect on either of the large conductance (250 pS and 160 pS) calcium-activated potassium channels in VMHN neurones. 6. Intracellular recordings were made from glucose-receptive VMHN neurones in rat brain slices. Cromakalim (50 microM) or diazoxide (60 microM) did not alter the firing rate or passive membrane properties of these neurones demonstrated to be sensitive to tolbutamide (0.1 mM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1467829

  8. 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

  9. Sodium and potassium competition in potassium-selective and non-selective channels

    NASA Astrophysics Data System (ADS)

    Sauer, David B.; Zeng, Weizhong; Canty, John; Lam, Yeeling; Jiang, Youxing

    2013-11-01

    Potassium channels selectively conduct K+, primarily to the exclusion of Na+, despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na+ and K+ binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K+ occupancy within the filter upon Na+ competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K+-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K+ sites. A double-barrier mechanism is proposed to explain K+ channel selectivity at low K+ concentrations.

  10. Allosteric Voltage Gating of Potassium Channels I

    PubMed Central

    Horrigan, Frank T.; Cui, Jianmin; Aldrich, Richard W.

    1999-01-01

    Activation of large conductance Ca2+-activated K+ channels is controlled by both cytoplasmic Ca2+ and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca2+-activated K+ currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca2+ (<1 nM). In response to a voltage step, IK activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of IK relaxation [τ(IK)] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. τ(IK) increases weakly with voltage from −500 to −20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = −0.29 e). Similarly, the steady state GK–V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z ≅ 0.4 e) at more negative voltages, where Po = 10−5–10−6. These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca2+, this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C–C and O–O transitions, whereas the C–O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca2+-activated K+ channel voltage gating, but also have important implications for understanding the mechanism of Ca2+ sensitivity. PMID:10436003

  11. Permeation of ions across the potassium channel: Brownian dynamics studies.

    PubMed

    Chung, S H; Allen, T W; Hoyles, M; Kuyucak, S

    1999-11-01

    The physical mechanisms underlying the transport of ions across a model potassium channel are described. The shape of the model channel corresponds closely to that deduced from crystallography. From electrostatic calculations, we show that an ion permeating the channel, in the absence of any residual charges, encounters an insurmountable energy barrier arising from induced surface charges. Carbonyl groups along the selectivity filter, helix dipoles near the oval chamber, and mouth dipoles near the channel entrances together transform the energy barrier into a deep energy well. Two ions are attracted to this well, and their presence in the channel permits ions to diffuse across it under the influence of an electric field. Using Brownian dynamics simulations, we determine the magnitude of currents flowing across the channel under various conditions. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength causes a steady decrease in the channel conductance. The current also decreases systematically when the effective dielectric constant of the channel is lowered. The conductance with the optimal choice of dipoles reproduces the experimental value when the dielectric constant of the channel is assumed to be 60. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates from Ohm's law at a higher applied potential. The reversal potentials obtained with asymmetrical solutions are in agreement with those predicted by the Nernst equation. The conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings for the transport of ions across the potassium channels and membrane channels in general.

  12. 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

  13. Hyperoxia confers myocardial protection in mechanically ventilated rats through the generation of free radicals and opening of mitochondrial ATP-sensitive potassium channels.

    PubMed

    Colantuono, Giuseppe; Tiravanti, Edy Altea; Di Venosa, Nicola; Cazzato, Antonia; Rastaldo, Raffaella; Cagiano, Raffaele; D'Agostino, Donato; Federici, Antonio; Fiore, Tommaso

    2008-01-01

    1. One hour exposure to hyperoxia has been shown previously to limit a subsequent ischaemia-reperfusion injury in spontaneously breathing rats. We tested the cardioprotective effect of a shorter period of hyperoxia during mechanical ventilation and the possible contribution of reactive oxygen species (ROS) and mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels. 2. Mechanically ventilated rats were exposed to normoxia (Fi O2 = 0.3) or hyperoxia (Fi O2 = 1.0) for 30 min and pH, P CO2, PO2, heart rate, airway and blood pressure were measured at baseline and after 30 min mechanical ventilation. Isolated hearts were subsequently subjected to 30 min ischaemia and 120 min reperfusion. Infarct size and left ventricular end-diastolic pressure (LVEDP), developed pressure (LVDP) and coronary flow (CF) were measured. In order to investigate the role of ROS and KATP channels within the mechanism leading to cardioprotection, the free radical scavenger N-acetylcysteine (NAC; 150 mg/kg) was infused in mechanically ventilated rats and the KATP channel blockers glibenclamide (200 mmol/L) or 5-hydroxydecanoate (10 mmol/L) were infused in isolated hearts immediately before ischaemia. 3. No differences were detected in P CO2, pH, heart rate, airway and blood pressure between the groups. However, the PO2 in hyperoxic groups was significantly higher compared with that in normoxic groups (P < 0.01). After 30 min ischaemia, we found that hyperoxic preconditioning significantly improved CF (P < 0.01), LVDP (P < 0.01) and LVEDP (P < 0.01) and reduced the extent of infarct size in the reperfused heart compared with the normoxic group (P < 0.01). When rats were pretreated either with NAC before hyperoxic ventilation or with K(ATP) channel blockers before ischaemia, myocardial protection was abolished. 4. Hyperoxic mechanical ventilation, prior to ischaemia, reduces myocardial reperfusion injury. This is likely to occur through the induction of oxidative stress, which leads to myocyte

  14. 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.

  15. Tarantula toxins interacting with voltage sensors in potassium channels

    PubMed Central

    Swartz, Kenton J.

    2007-01-01

    Voltage-activated ion channels open and close in response to changes in membrane voltage, a process that is crucial for electrical signaling in the nervous system. The venom from many poisonous creatures contains a diverse array of small protein toxins that bind to voltage-activated channels and modify the gating mechanism. Hanatoxin and a growing number of related tarantula toxins have been shown to inhibit activation of voltage-activated potassium (Kv) channels by interacting with their voltage sensing domains. This review summarizes our current understanding of the mechanism by which these toxins alter gating, the location of the toxin receptor within Kv channels and the disposition of this receptor with respect to the lipid membrane. The conservation of tarantula toxin receptors among voltage-activated ion channels will also be discussed. PMID:17097703

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

    PubMed

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

    2014-06-05

    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.

  17. 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

  18. Taurine blocks ATP-sensitive potassium channels of rat skeletal muscle fibres interfering with the sulphonylurea receptor.

    PubMed

    Tricarico, D; Barbieri, M; Camerino, D C

    2000-06-01

    Taurine is a sulphonic aminoacid present in high amounts in various tissues including cardiac and skeletal muscles showing different properties such as antioxidative, antimyotonic and anti-schaemic effects. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction of the sulphonic aminoacid with several ion channels. Using the patch-clamp technique we studied the effects of taurine in rat skeletal muscle fibres on ATP-sensitive K(+) channel (K(ATP)) immediately after excision and on channels that underwent rundown. The cytoplasmic application of 20 mM of taurine reduced the K(ATP) current; this effect was reverted by washout of the drug solution. In this experimental condition the IC(50) was 20.1 mM. After rundown, taurine inhibited the K(ATP) current with similar efficacy. Competition experiments showed that taurine shifted the dose-response inhibition curve of glybenclamide to the left on the log-dose axis without significantly affecting those of ATP or Ca(2+) ion. Single channel recording revealed that taurine affects the close state of the channel prolonging it and reducing the bursts duration. Our data indicate that taurine inhibits the muscular K(ATP) channel interfering with the glybenclamide site on the sulphonylurea receptor of the channel or on the site allosterically coupled to it. During ischaemia and hypoxia, the skeletal and heart muscles undergo several changes; for example, the activation of K(ATP) channels and loss of the intracellular taurine content. The depletion of taurine during ischaemia would contribute to the early activation of K(ATP) channels and salvage the intracellular ATP content.

  19. Potassium channel openers are uncoupling protonophores: implication in cardioprotection.

    PubMed

    Holmuhamedov, Ekhson L; Jahangir, Arshad; Oberlin, Andrew; Komarov, Alexander; Colombini, Marco; Terzic, Andre

    2004-06-18

    Excessive build-up of mitochondrial protonic potential is harmful to cellular homeostasis, and modulation of inner membrane permeability a proposed countermeasure. Here, we demonstrate that structurally distinct potassium channel openers, diazoxide and pinacidil, facilitated transmembrane proton translocation generating H(+)-selective current through planar phospholipid membrane. Both openers depolarized mitochondria, activated state 4 respiration and reduced oxidative phosphorylation, recapitulating the signature of mitochondrial uncoupling. This effect was maintained in K(+)-free conditions and shared with the prototypic protonophore 2,4-dinitrophenol. Diazoxide, pinacidil and 2,4-dinitrophenol, but not 2,4-dinitrotoluene lacking protonophoric properties, preserved functional recovery of ischemic heart. The identified protonophoric property of potassium channel openers, thus, implicates a previously unrecognized component in their mechanism of cardioprotection.

  20. Perivascular adipose tissue, potassium channels, and vascular dysfunction.

    PubMed

    Tano, Jean-Yves; Schleifenbaum, Johanna; Gollasch, Maik

    2014-09-01

    Perivascular adipose tissue has been recognized unequivocally as a major player in the pathology of metabolic and cardiovascular diseases. Through its production of adipokines and the release of other thus far unidentified factors, this recently discovered adipose tissue modulates vascular regulation and the myogenic response. After the discovery of its ability to diminish the vessel's response to vasoconstrictors, a new paradigm established adipose-derived relaxing factor (ADRF) as a paracrine smooth muscle cells' potassium channel opener that could potentially help combat vascular dysfunction. This review will discuss the role of ADRF in vascular dysfunction in obesity and hypertension, the different potassium channels that can be activated by this factor, and describes new pharmacological tools that can mimic the ADRF effect and thus can be beneficial against vascular dysfunction in cardiovascular disease. © 2014 American Heart Association, Inc.

  1. Trafficking of Neuronal Two Pore Domain Potassium Channels

    PubMed Central

    Mathie, Alistair; Rees, Kathryn A; El Hachmane, Mickael F; Veale, Emma L

    2010-01-01

    The activity of two pore domain potassium (K2P) channels regulates neuronal excitability and cell firing. Post-translational regulation of K2P channel trafficking to the membrane controls the number of functional channels at the neuronal membrane affecting the functional properties of neurons. In this review, we describe the general features of K channel trafficking from the endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus then focus on established regulatory mechanisms for K2P channel trafficking. We describe the regulation of trafficking of TASK channels from the ER or their retention within the ER and consider the competing hypotheses for the roles of the chaperone proteins 14-3-3, COP1 and p11 in these processes and where these proteins bind to TASK channels. We also describe the localisation of TREK channels to particular regions of the neuronal membrane and the involvement of the TREK channel binding partners AKAP150 and Mtap2 in this localisation. We describe the roles of other K2P channel binding partners including Arf6, EFA6 and SUMO for TWIK1 channels and Vpu for TASK1 channels. Finally, we consider the potential importance of K2P channel trafficking in a number of disease states such as neuropathic pain and cancer and the protection of neurons from ischemic damage. We suggest that a better understanding of the mechanisms and regulations that underpin the trafficking of K2P channels to the plasma membrane and to localised regions therein may considerably enhance the probability of future therapeutic advances in these areas. PMID:21358977

  2. The voltage-gated potassium channels and their relatives.

    PubMed

    Yellen, Gary

    2002-09-05

    The voltage-gated potassium channels are the prototypical members of a family of membrane signalling proteins. These protein-based machines have pores that pass millions of ions per second across the membrane with astonishing selectivity, and their gates snap open and shut in milliseconds as they sense changes in voltage or ligand concentration. The architectural modules and functional components of these sophisticated signalling molecules are becoming clear, but some important links remain to be elucidated.

  3. Monte Carlo study of gating and selection in potassium channels

    NASA Astrophysics Data System (ADS)

    Andreucci, Daniele; Bellaveglia, Dario; Cirillo, Emilio N. M.; Marconi, Silvia

    2011-08-01

    The study of selection and gating in potassium channels is a very important issue in modern biology. Indeed, such structures are known in essentially all types of cells in all organisms where they play many important functional roles. The mechanism of gating and selection of ionic species is not clearly understood. In this paper we study a model in which gating is obtained via an affinity-switching selectivity filter. We discuss the dependence of selectivity and efficiency on the cytosolic ionic concentration and on the typical pore open state duration. We demonstrate that a simple modification in the way in which the selectivity filter is modeled yields larger channel efficiency.

  4. 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.

  5. 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

  6. Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

    PubMed

    Tian, Lijun; Jeffries, Owen; McClafferty, Heather; Molyvdas, Adam; Rowe, Iain C M; Saleem, Fozia; Chen, Lie; Greaves, Jennifer; Chamberlain, Luke H; Knaus, Hans-Guenther; Ruth, Peter; Shipston, Michael J

    2008-12-30

    Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

  7. 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

  8. Modeling diverse range of potassium channels with Brownian dynamics.

    PubMed Central

    Chung, Shin-Ho; Allen, Toby W; Kuyucak, Serdar

    2002-01-01

    Using the experimentally determined KcsA structure as a template, we propose a plausible explanation for the diversity of potassium channels seen in nature. A simplified model of KcsA is constructed from its atomic resolution structure by smoothing out the protein-water boundary and representing the atoms forming the channel protein as a homogeneous, low dielectric medium. The properties of the simplified and atomic-detail models, deduced from electrostatic calculations and Brownian dynamics simulations, are shown to be qualitatively similar. We then study how the current flowing across the simplified model channel changes as the shape of the intrapore region is modified. This is achieved by increasing the radius of the intracellular pore systematically from 1.5 to 5 A while leaving the dimensions of the selectivity filter and inner chamber unaltered. The strengths of the dipoles located near the entrances of the channel, the carbonyl groups lining the selectivity filter, and the helix macrodipoles are kept constant. The channel conductance increases steadily as the radius of the intracellular pore is increased. The rate-limiting step for both the outward and inward current is the time it takes for an ion to cross the residual energy barrier located in the intrapore region. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates slightly from Ohm's law at higher applied potentials. The nonlinearity in the current-voltage curve becomes less pronounced as the radius of the intracellular pore is increased. When the strengths of the dipoles near the intracellular entrance are reduced, the channel shows a pronounced inward rectification. Finally, the conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings on the transport of ions across the potassium channels and membrane channels in general. PMID:12080118

  9. 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.

  10. Properties of shaker-type potassium channels in higher plants.

    PubMed

    Gambale, F; Uozumi, N

    2006-03-01

    Potassium (K(+)), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na(+)/K(+) exchanger, which widely exists in animal cells, K(+) channels and some type of K(+) transporters function as K(+) uptake systems in plants. Plant voltage-dependent K(+) channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K(+) channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K(+) channels have been identified and play a crucial role in K(+) homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K(+) channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K(+) channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K(+) channels in plants in comparison to those of Shaker channels in animals and bacteria.

  11. Engineering of an Artificial Light-Modulated Potassium Channel

    PubMed Central

    Caro, Lydia N.; Moreau, Christophe J.; Estrada-Mondragón, Argel; Ernst, Oliver P.; Vivaudou, Michel

    2012-01-01

    Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1F137S (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools. PMID:22928030

  12. Engineering of an artificial light-modulated potassium channel.

    PubMed

    Caro, Lydia N; Moreau, Christophe J; Estrada-Mondragón, Argel; Ernst, Oliver P; Vivaudou, Michel

    2012-01-01

    Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.

  13. Mechanism of electromechanical coupling in voltage-gated potassium channels.

    PubMed

    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 gating.

  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. Transmembrane allosteric coupling of the gates in a potassium channel

    PubMed Central

    Wylie, Benjamin J.; Bhate, Manasi P.; McDermott, Ann E.

    2014-01-01

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening. PMID:24344306

  16. Transmembrane allosteric coupling of the gates in a potassium channel.

    PubMed

    Wylie, Benjamin J; Bhate, Manasi P; McDermott, Ann E

    2014-01-07

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.

  17. Protein complex analysis of native brain potassium channels by proteomics.

    PubMed

    Sandoz, Guillaume; Lesage, Florian

    2008-01-01

    TREK potassium channels belong to a family of channel subunits with two-pore domains (K(2P)). TREK1 knockout mice display impaired polyunsaturated fatty acid-mediated protection against brain ischemia, reduced sensitivity to volatile anesthetics, resistance to depression and altered perception of pain. Recently, we isolated native TREK1 channels from mouse brain and identified their specific components by mass spectrometry. Among the identified partners, the A-Kinase Anchoring Protein AKAP150 binds to a regulatory domain of TREK1 and acts as a molecular switch. It transforms low activity, outwardly rectifying TREK1 currents into robust leak conductances resistant to stimulation by arachidonic acid, membrane stretch and acidification. Inhibition of the TREK1/AKAP150 channel by Gs-coupled receptors is as extensive as for TREK1 alone (but faster) whereas inhibition of TREK1/AKAP150 by Gq-coupled receptors is reduced. Furthermore, the association of AKAP150 with TREK1 channels integrates them into postsynaptic scaffolds where G protein-coupled membrane receptors and channels dock simultaneously. This chapter describes the proteomic approach used to study the composition of native TREK1 channels and point out its advantages and limitations over more classical methods (two-hybrid screenings in the yeast and bacteria or GST-pull down).

  18. Structural correlates of selectivity and inactivation in potassium channels

    PubMed Central

    McCoy, Jason G.; Nimigean, Crina M.

    2011-01-01

    Potassium channels are involved in a tremendously diverse range of physiological applications requiring distinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are diverse as well, except for the region that has been ordained the “selectivity filter”. The goal of this review is to examine our current understanding of the role of the selectivity filter and regions adjacent to it in specifying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in channels with the same signature sequence while at the same time affecting channel inactivation properties. PMID:21958666

  19. Quasi-specific access of the potassium channel inactivation gate.

    PubMed

    Venkataraman, Gaurav; Srikumar, Deepa; Holmgren, Miguel

    2014-06-09

    Many voltage-gated potassium channels open in response to membrane depolarization and then inactivate within milliseconds. Neurons use these channels to tune their excitability. In Shaker K(+) channels, inactivation is caused by the cytoplasmic amino terminus, termed the inactivation gate. Despite having four such gates, inactivation is caused by the movement of a single gate into a position that occludes ion permeation. The pathway that this single inactivation gate takes into its inactivating position remains unknown. Here we show that a single gate threads through the intracellular entryway of its own subunit, but the tip of the gate has sufficient freedom to interact with all four subunits deep in the pore, and does so with equal probability. This pathway demonstrates that flexibility afforded by the inactivation peptide segment at the tip of the N-terminus is used to mediate function.

  20. 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.

  1. Molecular basis of potassium channels in pancreatic duct epithelial cells.

    PubMed

    Hayashi, Mikio; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K(+) channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K(+) channels in pancreatic duct cells, including KCNN4 (KCa 3.1), KCNMA1 (KCa 1.1), KCNQ1 (Kv 7.1), KCNH2 (Kv 11.1), KCNH5 (Kv 10.2), KCNT1 (KCa 4.1), KCNT2 (KCa 4.2), and KCNK5 (K 2P 5.1). We will give an overview of K(+) channels with respect to their electrophysiological and pharmacological characteristics and regulation, which we know from other cell types, preferably in epithelia, and, where known, their identification and functions in pancreatic ducts and in adenocarcinoma cells. We conclude by pointing out some outstanding questions and future directions in pancreatic K(+) channel research with respect to the physiology of secretion and pancreatic pathologies, including pancreatitis, cystic fibrosis, and cancer, in which the dysregulation or altered expression of K(+) channels may be of importance.

  2. Molecular basis of potassium channels in pancreatic duct epithelial cells

    PubMed Central

    Hayashi, Mikio; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K+ channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K+ channels in pancreatic duct cells, including KCNN4 (KCa3.1), KCNMA1 (KCa1.1), KCNQ1 (Kv7.1), KCNH2 (Kv11.1), KCNH5 (Kv10.2), KCNT1 (KCa4.1), KCNT2 (KCa4.2), and KCNK5 (K2P5.1). We will give an overview of K+ channels with respect to their electrophysiological and pharmacological characteristics and regulation, which we know from other cell types, preferably in epithelia, and, where known, their identification and functions in pancreatic ducts and in adenocarcinoma cells. We conclude by pointing out some outstanding questions and future directions in pancreatic K+ channel research with respect to the physiology of secretion and pancreatic pathologies, including pancreatitis, cystic fibrosis, and cancer, in which the dysregulation or altered expression of K+ channels may be of importance. PMID:23962792

  3. 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).

  4. Imidazoline compounds stimulate insulin release by inhibition of K(ATP) channels and interaction with the exocytotic machinery.

    PubMed

    Zaitsev, S V; Efanov, A M; Efanova, I B; Larsson, O; Ostenson, C G; Gold, G; Berggren, P O; Efendić, S

    1996-11-01

    A novel imidazoline compound, RX871024, was used to investigate the mechanisms by which imidazoline derivatives promote insulin secretion in rat pancreatic beta-cells and HIT T15 cells. RX871024 stimulated insulin release from rat pancreatic beta-cells and HIT T15 cells in a glucose-dependent way. This effect was not related to alpha2-adrenergic, I1-, and I2-imidazoline receptors. RX871024 promoted insulin release by at least two modes of action. One included an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), subsequent to blocking of ATP-dependent K+ channels, membrane depolarization, and activation of voltage-dependent Ca2+ channels. The other, a more distal effect of imidazoline, affected the exocytotic machinery and was unrelated to changes in membrane potential and [Ca2+]i. The mechanism of RX871024-induced insulin release was dependent on protein kinases A and C. The sensitizing effect of a low dose of RX871024 on glucose-induced insulin secretion suggests that imidazoline compounds of this kind may constitute the basis for development of a new class of oral hypoglycemic agents.

  5. Potassium secretion in rat distal colon during dietary potassium loading: role of pH regulated apical potassium channels

    PubMed Central

    Sandle, G; Butterfield, I

    1999-01-01

    +-H+ exchange, which may be a manifestation of the secondary hyperaldosteronism associated with this model of colonic K+ adaptation. 

 Keywords: colon; dietary potassium; pH; potassium channels PMID:9862824

  6. Molecular mechanism of voltage sensor movements in a potassium channel.

    PubMed

    Elliott, David J S; Neale, Edward J; Aziz, Qadeer; Dunham, James P; Munsey, Tim S; Hunter, Malcolm; Sivaprasadarao, Asipu

    2004-12-08

    Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization--this motion is accompanied by a reorientation of S4 charges to the extracellular phase.

  7. Structural properties of PAS domains from the KCNH potassium channels.

    PubMed

    Adaixo, Ricardo; Harley, Carol A; Castro-Rodrigues, Artur F; Morais-Cabral, João H

    2013-01-01

    KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.

  8. 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.

  9. Reactive oxygen species and small-conductance calcium-dependent potassium channels are key mediators of inflammation-induced hypotension and shock

    PubMed Central

    Rogge, Elke; Janssen, Ben; Brouckaert, Peter

    2010-01-01

    Septic shock is associated with life-threatening vasodilation and hypotension. To cause vasodilation, vascular endothelium may release nitric oxide (NO), prostacyclin (PGI2), and the elusive endothelium-derived hyperpolarizing factor (EDHF). Although NO is critical in controlling vascular tone, inhibiting NO in septic shock does not improve outcome, on the contrary, precipitating the search for alternative therapeutic targets. Using a hyperacute tumor necrosis factor (TNF)-induced shock model in mice, we found that shock can develop independently of the known vasodilators NO, cGMP, PGI2, or epoxyeicosatrienoic acids. However, the antioxidant tempol efficiently prevented hypotension, bradycardia, hypothermia, and mortality, indicating the decisive involvement of reactive oxygen species (ROS) in these phenomena. Also, in classical TNF or lipopolysaccharide-induced shock models, tempol protected significantly. Experiments with (cell-permeable) superoxide dismutase or catalase, N-acetylcysteine and apocynin suggest that the ROS-dependent shock depends on intracellular \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ ^\\bullet {\\hbox{OH}} $$\\end{document} radicals. Potassium channels activated by ATP (KATP) or calcium (KCa) are important mediators of vascular relaxation. While NO and PGI2-induced vasodilation involves KATP and large-conductance BKCa channels, small-conductance SKCa channels mediate vasodilation induced by EDHF. Interestingly, also SKCa inhibition completely prevented the ROS-dependent shock. Our data thus indicate that intracellular \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin

  10. Chronic fluoxetine treatment increases NO bioavailability and calcium-sensitive potassium channels activation in rat mesenteric resistance arteries.

    PubMed

    Pereira, Camila A; Ferreira, Nathanne S; Mestriner, Fabiola L; Antunes-Rodrigues, José; Evora, Paulo R B; Resstel, Leonardo B M; Carneiro, Fernando S; Tostes, Rita C

    2015-10-15

    Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10 mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

  11. A large-conductance calcium-activated potassium channel in potato (Solanum tuberosum) tuber mitochondria.

    PubMed

    Koszela-Piotrowska, Izabela; Matkovic, Karolina; Szewczyk, Adam; Jarmuszkiewicz, Wieslawa

    2009-11-11

    In the present study, we describe the existence of a novel potassium channel in the plant [potato (Solanum tuberosum) tuber] mitochondrial inner membrane. We found that substances known to modulate large-conductance calcium-activated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca2+ and NS1619 {1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-ben-zimidazole-2-one; a potassium channel opener} were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilayers. After incorporation in 50/450 mM KCl gradient solutions, we recorded large-conductance potassium channel activity with conductance from 502+/-15 to 615+/-12 pS. The probability of channel opening was increased by Ca2+ and reduced by iberiotoxin. Immunological analysis with antibodies raised against the mammalian plasma-membrane large-conductance Ca2+-dependent K+ channel identified a pore-forming alpha subunit and an auxiliary beta2 subunit of the channel in potato tuber mitochondrial inner membrane. These results suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

  12. Cardiac potassium channel dysfunction in sudden infant death syndrome.

    PubMed

    Rhodes, Troy E; Abraham, Robert L; Welch, Richard C; Vanoye, Carlos G; Crotti, Lia; Arnestad, Marianne; Insolia, Roberto; Pedrazzini, Matteo; Ferrandi, Chiara; Vege, Ashild; Rognum, Torleiv; Roden, Dan M; Schwartz, Peter J; George, Alfred L

    2008-03-01

    Life-threatening arrhythmias have been suspected as one cause of the sudden infant death syndrome (SIDS), and this hypothesis is supported by the observation that mutations in arrhythmia susceptibility genes occur in 5-10% of cases. However, the functional consequences of cardiac potassium channel gene mutations associated with SIDS and how these alleles might mechanistically predispose to sudden death are unknown. To address these questions, we studied four missense KCNH2 (encoding HERG) variants, one compound KCNH2 genotype, and a missense KCNQ1 mutation all previously identified in Norwegian SIDS cases. Three of the six variants exhibited functional impairments while three were biophysically similar to wild-type channels (KCNH2 variants V279M, R885C, and S1040G). When co-expressed with WT-HERG, R273Q and K897T/R954C generated currents resembling the rapid component of the cardiac delayed rectifier current (I(Kr)) but with significantly diminished amplitude. Action potential modeling demonstrated that this level of functional impairment was sufficient to evoke increased action potential duration and pause-dependent early afterdepolarizations. By contrast, KCNQ1-I274V causes a gain-of-function in I(Ks) characterized by increased current density, faster activation, and slower deactivation leading to accumulation of instantaneous current upon repeated stimulation. Action potential simulations using a Markov model of heterozygous I274V-I(Ks) incorporated into the Luo-Rudy (LRd) ventricular cell model demonstrated marked rate-dependent shortening of action potential duration predicting a short QT phenotype. Our results indicate that certain potassium channel mutations associated with SIDS confer overt functional defects consistent with either LQTS or SQTS, and further emphasize the role of congenital arrhythmia susceptibility in this syndrome.

  13. 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

  14. Kv3.3 potassium channels and spinocerebellar ataxia.

    PubMed

    Zhang, Yalan; Kaczmarek, Leonard K

    2016-08-15

    The voltage-dependent potassium channel subunit Kv3.3 is expressed at high levels in cerebellar Purkinje cells, in auditory brainstem nuclei and in many other neurons capable of firing at high rates. In the cerebellum, it helps to shape the very characteristic complex spike of Purkinje cells. Kv3.3 differs from other closely related channels in that human mutations in the gene encoding Kv3.3 (KCNC3) result in a unique neurodegenerative disease termed spinocerebellar ataxia type 13 (SCA13). This primarily affects the cerebellum, but also results in extracerebellar symptoms. Different mutations produce either early onset SCA13, associated with delayed motor and impaired cognitive skill acquisition, or late onset SCA13, which typically produces cerebellar degeneration in middle age. This review covers the localization and physiological function of Kv3.3 in the central nervous system and how the normal function of the channel is altered by the disease-causing mutations. It also describes experimental approaches that are being used to understand how Kv3.3 mutations are linked to neuronal survival, and to develop strategies for treatment.

  15. Modulation of Potassium Channel Function by Methionine Oxidation and Reduction

    NASA Astrophysics Data System (ADS)

    Ciorba, Matthew A.; Heinemann, Stefan H.; Weissbach, Herbert; Brot, Nathan; Hoshi, Toshinori

    1997-09-01

    Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.

  16. The renal TRPV4 channel is essential for adaptation to increased dietary potassium.

    PubMed

    Mamenko, Mykola V; Boukelmoune, Nabila; Tomilin, Viktor N; Zaika, Oleg L; Jensen, V Behrana; O'Neil, Roger G; Pochynyuk, Oleh M

    2017-06-01

    To maintain potassium homeostasis, kidneys exert flow-dependent potassium secretion to facilitate kaliuresis in response to elevated dietary potassium intake. This process involves stimulation of calcium-activated large conductance maxi-K (BK) channels in the distal nephron, namely the connecting tubule and the collecting duct. Recent evidence suggests that the TRPV4 channel is a critical determinant of flow-dependent intracellular calcium elevations in these segments of the renal tubule. Here, we demonstrate that elevated dietary potassium intake (five percent potassium) increases renal TRPV4 mRNA and protein levels in an aldosterone-dependent manner and causes redistribution of the channel to the apical plasma membrane in native collecting duct cells. This, in turn, leads to augmented TRPV4-mediated flow-dependent calcium ion responses in freshly isolated split-opened collecting ducts from mice fed the high potassium diet. Genetic TRPV4 ablation greatly diminished BK channel activity in collecting duct cells pointing to a reduced capacity to excrete potassium. Consistently, elevated potassium intake induced hyperkalemia in TRPV4 knockout mice due to deficient renal potassium excretion. Thus, regulation of TRPV4 activity in the distal nephron by dietary potassium is an indispensable component of whole body potassium balance. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. 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.

  18. 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

    2015-01-06

    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.

  19. Depletion of intracellular polyamines relieves inward rectification of potassium channels.

    PubMed

    Shyng, S L; Sha, Q; Ferrigni, T; Lopatin, A N; Nichols, C G

    1996-10-15

    Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches.

  20. Nitric oxide inhibits irreversibly P815 cell proliferation: involvement of potassium channels.

    PubMed

    Costa, R S A; Assreuy, J

    2002-12-01

    Nitric oxide (NO) has been shown to inhibit both normal and cancer cell proliferation. Potassium channels are involved in cell proliferation and, as NO activates these channels, we investigated the effect of NO on the proliferation of murine mastocytoma cell lines and the putative involvement of potassium channels. NO (in the form of NO donors) caused dose-dependent inhibition of cell proliferation in the P815 cell line inducing growth arrest in the mitosis phase. Incubation with NO donor for 4 or 24 h had a similar inhibitory effect on cell proliferation, indicating that this effect is irreversible. The inhibitory effect of NO was completely prevented by the blockade of voltage- and calcium-dependent potassium channels, but not by blockade of ATP-dependent channels. NO inhibition of cell proliferation was unaffected by guanylate cyclase and by cytoskeleton disruptors. Therefore, NO inhibits cell proliferation irreversibly via a potassium channel-dependent but guanylate cyclase-independent pathway in murine mastocytoma cells.

  1. Cilostazol protects the heart against ischaemia reperfusion injury in a rabbit model of myocardial infarction: focus on adenosine, nitric oxide and mitochondrial ATP-sensitive potassium channels.

    PubMed

    Bai, Yushan; Muqier; Murakami, Hiroya; Iwasa, Masamitsu; Sumi, Shohei; Yamada, Yoshihisa; Ushikoshi, Hiroaki; Aoyama, Takuma; Nishigaki, Kazuhiko; Takemura, Genzou; Uno, Bunji; Minatoguchi, Shinya

    2011-10-01

    1. The present study examined whether or not cilostazol reduces the myocardial infarct size, and investigated its mechanism in a rabbit model of myocardial infarction. 2. Japanese white rabbits underwent 30 min of coronary occlusion, followed by 48 h of reperfusion. Cilostazol (1 and 5 mg/kg) or vehicle was given intravenously 5 min before ischaemia. 8-p-sulfophenyl theophylline (8SPT; an adenosine receptor blocker, 7.5 mg/kg), Nω-nitro-L-arginine methylester (l-NAME; an NOS inhibitor, 10 mg/kg) or 5-hydroxydecanoic acid sodium salt (5-HD; a mitochondrial ATP-sensitive potassium (KATP) channel blocker, 5 mg/kg) was given intravenously 5 min before cilostazol injection. Infarct size was determined as a percentage of the risk area. 3. The myocardial interstitial levels of adenosine and nitrogen oxide (NOx) during ischaemia and reperfusion, and the intensity of myocardial dihydroethidium staining were determined. 4. Infarct size was significantly reduced in the cilostazol 1 mg/kg (38.4% (2.9%)) and cilostazol 5 mg/kg (30.7% (4.7%)) groups compared with that in the control group (46.5% (4.2%)). The infarct size-reducing effect of cilostazol was completely abolished by 8SPT (46.6% (3.5%)), L-NAME (49.0% (5.5%)), or 5HD (48.5% (5.1%)). 8SPT, L-NAME or 5HD alone did not affect the infarct size. Cilostazol treatment significantly increased myocardial levels of adenosine and NOx during ischaemia, and attenuated the intensity of dihydroethidium staining during reperfusion. 5. These findings show that cilostazol reduces the myocardial infarct size by increasing adenosine and NOx levels, attenuating superoxide production and opening the mitochondrial KATP channels. Cilostazol might provide a new strategy for the treatment of coronary heart disease.

  2. Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons

    PubMed Central

    Bock, Tobias

    2016-01-01

    Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels on N-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive. PMID:26936985

  3. Clustering of neuronal potassium channels is independent of their interaction with PSD-95

    PubMed Central

    Rasband, Matthew N.; Park, Eunice W.; Zhen, Dongkai; Arbuckle, Margaret I.; Poliak, Sebastian; Peles, Elior; Grant, Seth G.N.; Trimmer, James S.

    2002-01-01

    Voltage-dependent potassium channels regulate membrane excitability and cell–cell communication in the mammalian nervous system, and are found highly localized at distinct neuronal subcellular sites. Kv1 (mammalian Shaker family) potassium channels and the neurexin Caspr2, both of which contain COOH-terminal PDZ domain binding peptide motifs, are found colocalized at high density at juxtaparanodes flanking nodes of Ranvier of myelinated axons. The PDZ domain–containing protein PSD-95, which clusters Kv1 potassium channels in heterologous cells, has been proposed to play a major role in potassium channel clustering in mammalian neurons. Here, we show that PSD-95 colocalizes precisely with Kv1 potassium channels and Caspr2 at juxtaparanodes, and that a macromolecular complex of Kv1 channels and PSD-95 can be immunopurified from mammalian brain and spinal cord. Surprisingly, we find that the high density clustering of Kv1 channels and Caspr2 at juxtaparanodes is normal in a mutant mouse lacking juxtaparanodal PSD-95, and that the indirect interaction between Kv1 channels and Caspr2 is maintained in these mutant mice. These data suggest that the primary function of PSD-95 at juxtaparanodes lies outside of its accepted role in mediating the high density clustering of Kv1 potassium channels at these sites. PMID:12438413

  4. Opening of the mitoKATP channel and decoupling of mitochondrial complex II and III contribute to the suppression of myocardial reperfusion hyperoxygenation.

    PubMed

    Liu, Bin; Zhu, Xuehai; Chen, Chwen-Lih; Hu, Keli; Swartz, Harold M; Chen, Yeong-Renn; He, Guanglong

    2010-04-01

    Diazoxide, a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener, protects the heart from ischemia-reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O(2)(-*)) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O(2)(-*) generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV-visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoK(ATP) channel and ubisemiquinone-dependent O(2)(-*) generation via inhibiting mitochondrial complex II-dependent respiration.

  5. A comprehensive guide to the ROMK potassium channel: form and function in health and disease

    PubMed Central

    2009-01-01

    The discovery of the renal outer medullary K+ channel (ROMK, Kir1.1), the founding member of the inward-rectifying K+ channel (Kir) family, by Ho and Hebert in 1993 revolutionized our understanding of potassium channel biology and renal potassium handling. Because of the central role that ROMK plays in the regulation of salt and potassium homeostasis, considerable efforts have been invested in understanding the underlying molecular mechanisms. Here we provide a comprehensive guide to ROMK, spanning from the physiology in the kidney to the organization and regulation by intracellular factors to the structural basis of its function at the atomic level. PMID:19458126

  6. 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

  7. 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.

  8. Cell-based potassium ion channel screening using the FluxOR assay.

    PubMed

    Beacham, Daniel W; Blackmer, Trillium; O' Grady, Michael; Hanson, George T

    2010-04-01

    FluxOR technology is a cell-based assay used for high-throughput screening measurements of potassium channel activity. Using thallium influx as a surrogate indicator of potassium ion channel activity, the FluxOR Potassium Ion Channel Assay is based on the activation of a novel fluorescent dye. This indicator reports channel activity with a large fluorogenic response and is proportional to the number of open potassium channels on the cell, making it extremely useful for studying K(+) channel targets. In contrast to BTC-AM ester, FluxOR dye is roughly 10-fold more thallium sensitive, requiring much lower thallium for a larger signal window. This also means that the assay is carried out in a physiological, normal-chloride saline. In this article, the authors describe how they used BacMam gene delivery to express Kv7.2 and 7.3 (KCNQ), Kir2.1, or Kv11.1 (hERG) potassium ion channels in U2-OS cells. Using these cells, they ran the FluxOR assay to identify and characterize channel-specific inhibitory compounds discovered within the library (Tocriscreen Mini 1200 and Sigma Sodium/Potassium Modulators Ligand set). The FluxOR assay was able to identify several known specific inhibitors of Kv7.2/7.3 or hERG, highlighting its potential to identify novel and more efficacious small-molecule modulators.

  9. 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.

  10. Model Development for the Viral Kcv Potassium Channel

    PubMed Central

    Tayefeh, Sascha; Kloss, Thomas; Kreim, Michael; Gebhardt, Manuela; Baumeister, Dirk; Hertel, Brigitte; Richter, Christian; Schwalbe, Harald; Moroni, Anna; Thiel, Gerhard; Kast, Stefan M.

    2009-01-01

    Abstract A computational model for the open state of the short viral Kcv potassium channel was created and tested based on homology modeling and extensive molecular-dynamics simulation in a membrane environment. Particular attention was paid to the structure of the highly flexible N-terminal region and to the protonation state of membrane-exposed lysine residues. Data from various experimental sources, NMR spectroscopy, and electrophysiology, as well as results from three-dimensional reference interaction site model integral equation theory were taken into account to select the most reasonable model among possible variants. The final model exhibits spontaneous ion transitions across the complete pore, with and without application of an external field. The nonequilibrium transport events could be induced reproducibly without abnormally large driving potential and without the need to place ions artificially at certain key positions along the transition path. The transport mechanism through the filter region corresponds to the classic view of single-file motion, which in our case is coupled to frequent exchange of ions between the innermost filter position and the cavity. PMID:19167299

  11. 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

  12. 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

  13. Calcium and Potassium Channels in Experimental Subarachnoid Hemorrhage and Transient Global Ischemia

    PubMed Central

    Kamp, Marcel A.; Dibué, Maxine; Schneider, Toni; Steiger, Hans-Jakob; Hänggi, Daniel

    2012-01-01

    Healthy cerebrovascular myocytes express members of several different ion channel families which regulate resting membrane potential, vascular diameter, and vascular tone and are involved in cerebral autoregulation. In animal models, in response to subarachnoid blood, a dynamic transition of ion channel expression and function is initiated, with acute and long-term effects differing from each other. Initial hypoperfusion after exposure of cerebral vessels to oxyhemoglobin correlates with a suppression of voltage-gated potassium channel activity, whereas delayed cerebral vasospasm involves changes in other potassium channel and voltage-gated calcium channels expression and function. Furthermore, expression patterns and function of ion channels appear to differ between main and small peripheral vessels, which may be key in understanding mechanisms behind subarachnoid hemorrhage-induced vasospasm. Here, changes in calcium and potassium channel expression and function in animal models of subarachnoid hemorrhage and transient global ischemia are systematically reviewed and their clinical significance discussed. PMID:23251831

  14. Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

    PubMed

    Levitz, Joshua; Royal, Perrine; Comoglio, Yannick; Wdziekonski, Brigitte; Schaub, Sébastien; Clemens, Daniel M; Isacoff, Ehud Y; Sandoz, Guillaume

    2016-04-12

    Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

  15. 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…

  16. 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…

  17. Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A

    PubMed Central

    Salman, Shaima; Holloway, Alison C.

    2014-01-01

    At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K+ (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved μ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined μ {[d-Arg2,Ly4]dermorphin-(1–4)-amide}- and δ ([d-Pen2,5,P-Cl-Phe4]enkephalin)-opioid agonists (2 μM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers. PMID:24898587

  18. Psychiatric presentation of voltage-gated potassium channel antibody-associated encephalopathy. Case report.

    PubMed

    Parthasarathi, U D; Harrower, T; Tempest, M; Hodges, J R; Walsh, C; McKenna, P J; Fletcher, P C

    2006-08-01

    Voltage-gated potassium channel antibody encephalopathy, a rare cause of limbic encephalopathy, typically presents with memory impairment and seizures. Psychiatric symptoms have not been emphasised in the literature. Here we describe a 58-year-old man who presented with panic attacks and psychogenic non-epileptic seizures and, later on, developed delusions and hallucinations and then confusion. He was found to have antibodies to voltage-gated potassium channels. Treatment with immuno-modulatory therapy resulted in almost complete recovery.

  19. Different calcium channels are coupled to potassium channels with distinct physiological roles in vagal neurons.

    PubMed

    Sah, P

    1995-04-22

    Whole-cell and sharp microelectrode recordings were obtained from neurons of rat dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. Calcium currents were studied with sodium currents blocked with tetrodotoxin, potassium currents blocked by perfusing the cell with caesium as the main cation and using barium as the charge carrier. From a holding potential of -60 mV, inward currents activated at potentials positive of -50 mV and peaked around 0 mV. Voltage clamping the neuron at more hyperpolarised potentials did not reveal any low-threshold inward current. The inward current was effectively blocked by cadmium (100 microM) and nicked (1 mM), suggesting that it is carried by voltage-dependent calcium channels. The inward current could be separated into three pharmacologically distinct components: 40% of the whole cell current was omega-conotoxin sensitive; 20% of the current was nifedipine sensitive; and the rest was blocked by high concentrations of cadmium and nickel. This remaining current cannot be due to P-type calcium channels as omega-agatoxin had no effect on the inward current. Nifedipine had no significant effect on the action potential. Application of omega-conotoxin reduced the calcium component of the action potential and significantly reduced the potassium current underlying the afterhyperpolarization. Application of charybdotoxin slowed action potential repolarization. When N-type calcium channels were blocked with omega-conotoxin, charybdotoxin was still effective in slowing repolarization. In contrast, charybdotoxin was ineffective ineffective when calcium influx was blocked with the non-specific calcium channel blocker cadmium.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. TbIRK is a signature sequence free potassium channel from Trypanosoma brucei locating to acidocalcisomes.

    PubMed

    Steinmann, Michael E; Schmidt, Remo S; Bütikofer, Peter; Mäser, Pascal; Sigel, Erwin

    2017-04-06

    Potassium channels from prokaryotes and eukaryotes are usually recognized by a typical amino acid sequence TXTGY(F)G representing the ionic selectivity filter. Using a screening approach with ion channel family profiles but without the above motif, we identified a gene in Trypanosoma brucei that exhibits homology to inward rectifying potassium channels. We report here cloning of this ion channel named TbIRK. The protein is localized to acidocalcisomes in procyclic and in bloodstream form parasites. Functional properties of this channel were established after expression in Xenopus oocytes. Currents recorded in potassium medium show inward rectification and little time dependence. Surprisingly, this channel retains selectivity for potassium ions over sodium ions >7, in spite of the lack of the classical selectivity filter. The sequence GGYVG was predicted in silico to replace this filter motif. Point mutations of the corresponding glycine residues confirmed this at the functional level. The channel is inhibited by caesium ions but remains unaffected by barium ions up to 10 mM. TbIRK is to our knowledge the first potassium channel in T. brucei that localizes to the acidocalcisomes, organelles involved in the storage of phosphates and the response to osmotic stress that occurs during the life cycle of trypanosomes.

  1. Endocytic regulation of voltage-dependent potassium channels in the heart.

    PubMed

    Ishii, Kuniaki; Norota, Ikuo; Obara, Yutaro

    2012-01-01

    Understanding the regulation of cardiac ion channels is critical for the prevention of arrhythmia caused by abnormal excitability. Ion channels can be regulated by a change in function (qualitative) and a change in number (quantitative). Functional changes have been extensively investigated for many ion channels including cardiac voltage-dependent potassium channels. By contrast, the regulation of ion channel numbers has not been widely examined, particularly with respect to acute modulation of ion channels. This article briefly summarizes stimulus-induced endocytic regulation of major voltage-dependent potassium channels in the heart. The stimuli known to cause their endocytosis include receptor activation, drugs, and low extracellular [K(+)], following which the potassium channels undergo either clathrin-mediated or caveolin-mediated endocytosis. Receptor-mediated endocytic regulation has been demonstrated for Kv1.2, Kv1.5, KCNQ1 (Kv7.1), and Kv4.3, while drug-induced endocytosis has been demonstrated for Kv1.5 and hERG. Low [K(+)](o)-induced endocytosis might be unique for hERG channels, whose electrophysiological characteristics are known to be under strong influence of [K(+)](o). Although the precise mechanisms have not been elucidated, it is obvious that major cardiac voltage-dependent potassium channels are modulated by endocytosis, which leads to changes in cardiac excitability.

  2. Potassium

    MedlinePlus

    ... the potassium you need. However, certain diseases (e.g., kidney disease and gastrointestinal disease with vomiting and ... substitute and to eat potassium-rich foods (e.g., bananas, prunes, raisins, and milk).

  3. Big Potassium (BK) ion channels in biology, disease and possible targets for cancer immunotherapy

    PubMed Central

    Ge, Lisheng; Hoa, Neil T.; Wilson, Zechariah; Arismendi-Morillo, Gabriel; Kong, Xia-Tang; Tajhya, Rajeev B.; Beeton, Christine; Jadus, Martin R.

    2017-01-01

    The Big Potassium (BK) ion channel is commonly known by a variety of names (Maxi-K, KCNMA1, slo, Stretch-activated potassium channels, KCa1.1). Each name reflects a different physical property displayed by this single ion channel. This transmembrane channel is found on nearly every cell type of the body and has its own distinctive roles for that tissue type. The BKα channel contains the pore that releases potassium ions from intracellular stores. This ion channel is found on the cell membrane, endoplasmic reticulum, Golgi and mitochondria. Complex splicing pathways produce different isoforms. The BKα channels can be phosphorylated, palmitoylated and myristylated. BK is composed of a homo-tetramer that interacts with β and γ chains. These accessory proteins provide a further modulating effect on the functions of BKα channels. BK channels play important roles in cell division and migration. In this review, we will focus on the biology of BK channels, especially its role, and that it has in the immune response towards cancer. Recent proteomic studies have linked BK channels with various proteins. Some of these interactions offer further insight into the role that BK channels have with cancers, especially with brain tumors. This review shows that BK channels have a complex interplay with intracellular components of cancer cells and still have plenty of secrets to be discovered. PMID:25027630

  4. 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

  5. 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.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

    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.

  8. Potassium ions in the cavity of a KcsA channel model.

    PubMed

    Yao, Zhenwei; Qiao, Baofu; Olvera de la Cruz, Monica

    2013-12-01

    The high rate of ion flux and selectivity of potassium channels has been attributed to the conformation and dynamics of the ions in the filter which connects the channel cavity and the extracellular environment. The cavity serves as the reservoir for potassium ions diffusing from the intracellular medium. The cavity is believed to decrease the dielectric barrier for the ions to enter the filter. We study here the equilibrium and dynamic properties of potassium ions entering the water-filled cavity of a KcsA channel model. Atomistic molecular dynamics simulations that are supplemented by electrostatic calculations reveal the important role of water molecules and the partially charged protein helices at the bottom of the cavity in overcoming the energy barrier and stabilizing the potassium ion in the cavity. We further show that the average time for a potassium ion to enter the cavity is much shorter than the conduction rate of a potassium passing through the filter, and this time duration is insensitive over a wide range of the membrane potential. The conclusions drawn from the study of the channel model are applicable in generalized contexts, including the entry of ions in artificial ion channels and other confined geometries.

  9. Potassium ions in the cavity of a KcsA channel model

    NASA Astrophysics Data System (ADS)

    Yao, Zhenwei; Qiao, Baofu; Olvera de la Cruz, Monica

    2013-12-01

    The high rate of ion flux and selectivity of potassium channels has been attributed to the conformation and dynamics of the ions in the filter which connects the channel cavity and the extracellular environment. The cavity serves as the reservoir for potassium ions diffusing from the intracellular medium. The cavity is believed to decrease the dielectric barrier for the ions to enter the filter. We study here the equilibrium and dynamic properties of potassium ions entering the water-filled cavity of a KcsA channel model. Atomistic molecular dynamics simulations that are supplemented by electrostatic calculations reveal the important role of water molecules and the partially charged protein helices at the bottom of the cavity in overcoming the energy barrier and stabilizing the potassium ion in the cavity. We further show that the average time for a potassium ion to enter the cavity is much shorter than the conduction rate of a potassium passing through the filter, and this time duration is insensitive over a wide range of the membrane potential. The conclusions drawn from the study of the channel model are applicable in generalized contexts, including the entry of ions in artificial ion channels and other confined geometries.

  10. 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).

  11. 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

  12. 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.

  13. Origins of open-channel noise in the large potassium channel of sarcoplasmic reticulum

    PubMed Central

    1994-01-01

    Open-channel noise was studied in the large potassium channel of the sarcoplasmic reticulum (SR). Inside-out patches were excised directly from the SR of split skeletal muscle fibers of lobster, with lobster relaxing ringer (LRR) in bath and pipette. The power spectrum of open- channel noise is very low and approximately flat in the 100 Hz-10 kHz frequency range. At 20 degrees C, with an applied voltage of 50 mV, the mean single-channel current (i) is 9 pA (mean single-channel conductance = 180 pS) and the mean power spectral density 1.1 x 10(-29) A2/Hz. The latter increases nonlinearly with (i), showing a progressively steeper dependence as (i) increases. At 20 mV, the mean power spectral density is almost independent of (i) and approximately 1.4 times that of the Johnson noise calculated for the equivalent ideal resistor with zero net current; at 70 mV it increases approximately in proportion to (i)2. The mean power spectral density has a weak temperature dependence, very similar to that of (i), and both are well described by a Q10 of 1.3 throughout the range 3-40 degrees C. Discrete ion transport events are thought to account for a significant fraction of the measured open-channel noise, probably approximately 30-50% at 50 mV. Brief interruptions of the single-channel current, due either to blockage of the open channel by an extrinsic aqueous species, or to intrinsic conformational changes in the channel molecule itself, were a possible additional source of open-channel noise. Experiments in modified bathing solutions indicate, however, that open-channel noise is not affected by any of the identified aqueous species present in LRR. In particular, magnesium ions, the species thought most likely to cause brief blockages, and calcium and hydrogen ions, have no detectable effect. This channel's openings exhibit many brief closings and substrates, due to intrinsic gating of the channel. Unresolved brief full closings are calculated to make a negligible contribution

  14. Arachidonic Acid and Other Fatty Acids Directly Activate Potassium Channels in Smooth Muscle Cells

    NASA Astrophysics Data System (ADS)

    Ordway, Richard W.; Walsh, John V.; Singer, Joshua J.

    1989-06-01

    Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.

  15. The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney

    PubMed Central

    Fang, Liang; Garuti, Rita; Kim, Bo-Young; Wade, James B.; Welling, Paul A.

    2009-01-01

    Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical “NPXY” endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis. PMID:19841541

  16. Molecular simulation of the interaction of kappa-conotoxin-PVIIA with the Shaker potassium channel pore.

    PubMed

    Moran, O

    2001-12-01

    Molecular simulation techniques were appplied to predict the interaction of the voltage-dependent Shaker potassium channel with the channel-blocking toxin kappa-conotoxin-PVIIA (PVIIA). A structural thee-dimensional model of the extracellular vestibule of the potassium channel was constructed based on structural homologies with the bacterial potassium channel Kcsa, whose structure has been solved by X-ray crystallography. The docking of the PVIIA molecule was obtained by a geometric recognition algorithm, yielding 100 possible conformations. A series of residue-residue distance restraints, predicted from mutation-cycle experiments, were used to select a small set of a plausible channel-toxin complex models among the resulting possible conformations. The four final conformations, with similar characteristics, can explain most of the single-point mutation experiments done with this system. The models of the Shaker-PVIIA interaction predict two clusters of amino acids, critical for the binding of the toxin to the channel. The first cluster is the amino acids R2, I3, Q6 and K7 that form the plug of the toxin that interacts with the entrance to the selectivity filter of the channel. The second cluster of residues, R22, F23, N24 and K25, interacts with a channel region near to the external entrance of the pore vestibule. The consistency of the obtained models and the experimental data indicate that the Shaker-PVIIA complex model is reasonable and can be used in further biological studies such as the rational design of blocking agents of potassium channels and the mutagenesis of both toxins and potassium channels.

  17. The Eag potassium channel as a new prognostic marker in ovarian cancer

    PubMed Central

    2010-01-01

    Background Ovarian cancer is the second most common cancer of the female genital tract in the United Kingdom (UK), accounting for 6% of female deaths due to cancer. This cancer is associated with poor survival and there is a need for new treatments in addition to existing chemotherapy to improve survival. Potassium (K+) channels have been shown to be overexpressed in various cancers where they appear to play a role in cell proliferation and progression. Objectives To determine the expression of the potassium channels Eag and HERG in ovarian cancer tissue and to assess their role in cell proliferation. Methods The expression of Eag and HERG potassium channels was examined in an ovarian cancer tissue microarray. Their role in cell proliferation was investigated by blocking voltage-gated potassium channels in an ovarian cancer cell line (SK-OV-3). Results We show for the first time that high expression of Eag channels in ovarian cancer patients is significantly associated with poor survival (P = 0.016) unlike HERG channel expression where there was no correlation with survival. There was also a significant association of Eag staining with high tumour grade (P = 0.014) and presence of residual disease (P = 0.011). Proliferation of SK-OV-3 cells was significantly (P < 0.001) inhibited after treatment with voltage gated K+ channel blockers. Conclusion This novel finding demonstrates a role for Eag as a prognostic marker for survival in patients with ovarian cancer. PMID:21138547

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

    PubMed

    Fowler, Philip W; Abad, Enrique; Beckstein, Oliver; Sansom, Mark S P

    2013-11-12

    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.

  19. Viruses infecting marine picoplancton encode functional potassium ion channels.

    PubMed

    Siotto, Fenja; Martin, Corinna; Rauh, Oliver; Van Etten, James L; Schroeder, Indra; Moroni, Anna; Thiel, Gerhard

    2014-10-01

    Phycodnaviruses are dsDNA viruses, which infect algae. Their large genomes encode many gene products, like small K(+) channels, with homologs in prokaryotes and eukaryotes. Screening for K(+) channels revealed their abundance in viruses from fresh-water habitats. Recent sequencing of viruses from marine algae or from salt water in Antarctica revealed sequences with the predicted characteristics of K(+) channels but with some unexpected features. Two genes encode either 78 or 79 amino acid proteins, which are the smallest known K(+) channels. Also of interest is an unusual sequence in the canonical α-helixes in K(+) channels. Structural prediction algorithms indicate that the new channels have the conserved α-helix folds but the algorithms failed to identify the expected transmembrane domains flanking the K(+) channel pores. In spite of these unexpected properties electophysiological studies confirmed that the new proteins are functional K(+) channels. Copyright © 2014. Published by Elsevier Inc.

  20. 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

  1. 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.

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

    PubMed Central

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

    2016-01-01

    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 martensii Karsch 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 luteus as 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 IC50 value 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 BmKDfsin4adopts 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. PMID:26817841

  3. 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. Published by Elsevier Inc.

  4. Grafting voltage and pharmacological sensitivity in potassium channels

    PubMed Central

    Lan, Xi; Fan, Chunyan; Ji, Wei; Tian, Fuyun; Xu, Tao; Gao, Zhaobing

    2016-01-01

    A classical voltage-gated ion channel consists of four voltage-sensing domains (VSDs). However, the roles of each VSD in the channels remain elusive. We developed a GVTDT (Graft VSD To Dimeric TASK3 channels that lack endogenous VSDs) strategy to produce voltage-gated channels with a reduced number of VSDs. TASK3 channels exhibit a high host tolerance to VSDs of various voltage-gated ion channels without interfering with the intrinsic properties of the TASK3 selectivity filter. The constructed channels, exemplified by the channels grafted with one or two VSDs from Kv7.1 channels, exhibit classical voltage sensitivity, including voltage-dependent opening and closing. Furthermore, the grafted Kv7.1 VSD transfers the potentiation activity of benzbromarone, an activator that acts on the VSDs of the donor channels, to the constructed channels. Our study indicates that one VSD is sufficient to voltage-dependently gate the pore and provides new insight into the roles of VSDs. PMID:27174053

  5. Simulations of ion current in realistic models of ion channels: the KcsA potassium channel.

    PubMed

    Burykin, A; Schutz, C N; Villá, J; Warshel, A

    2002-05-15

    Realistic studies of ion current in biologic channels present a major challenge for computer simulation approaches. All-atom molecular dynamics simulations involve serious time limitations that prevent their use in direct evaluation of ion current in channels with significant barriers. The alternative use of Brownian dynamics (BD) simulations can provide the current for simplified macroscopic models. However, the time needed for accurate calculations of electrostatic energies can make BD simulations of ion current expensive. The present work develops an approach that overcomes some of the above challenges and allows one to simulate ion currents in models of biologic channels. Our method provides a fast and reliable estimate of the energetics of the system by combining semimacroscopic calculations of the self-energy of each ion and an implicit treatment of the interactions between the ions, as well as the interactions between the ions and the protein-ionizable groups. This treatment involves the use of the semimacroscopic version of the protein dipole Langevin dipole (PDLD/S) model in its linear response approximation (LRA) implementation, which reduces the uncertainties about the value of the protein "dielectric constant." The resulting free energy surface is used to generate the forces for on-the-fly BD simulations of the corresponding ion currents. Our model is examined in a preliminary simulation of the ion current in the KcsA potassium channel. The complete free energy profile for a single ion transport reflects reasonable energetics and captures the effect of the protein-ionized groups. This calculated profile indicates that we are dealing with the channel in its closed state. Reducing the barrier at the gate region allows us to simulate the ion current in a reasonable computational time. Several limiting cases are examined, including those that reproduce the observed current, and the nature of the productive trajectories is considered. The ability to simulate

  6. Potassium channels in the heart: structure, function and regulation.

    PubMed

    Grandi, Eleonora; Sanguinetti, Michael C; Bartos, Daniel C; Bers, Donald M; Chen-Izu, Ye; Chiamvimonvat, Nipavan; Colecraft, Henry M; Delisle, Brian P; Heijman, Jordi; Navedo, Manuel F; Noskov, Sergei; Proenza, Catherine; Vandenberg, Jamie I; Yarov-Yarovoy, Vladimir

    2017-04-01

    This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was 'K(+) Channels and Regulation'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K(+) channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage-gated K(+) channel function, as well as the mechanisms involved in regulation of K(+) channel gating, expression and membrane localization. Given the critical role for K(+) channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K(+) channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K(+) channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K(+) channels in cardiac electrophysiology, i.e. how K(+) currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K(+) channel-based therapeutics. A fundamental understanding of K(+) channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.

  7. Effect of lanthanum on voltage-dependent gating of a cloned mammalian neuronal potassium channel.

    PubMed

    Tytgat, J; Daenens, P

    1997-02-28

    The effect of the trivalent cation lanthanum (La3+) on voltage-dependent gating of a cloned mammalian neuronal Kv1.1 potassium channel was studied under whole-cell voltage-clamp conditions in oocytes of Xenopus laevis. La3+ (100 microM) was found to decrease the potassium currents at all test potentials and to shift the midpoint of the fraction open channels/membrane voltage curve by approximately +20 mV. The opening and closing time constants of Kv1.1 channels were empirically fitted with a 4th power Hodgkin-Huxley formalism, or with mono- and multi-exponentials. It was found that La3+ slowed down the kinetics of activation, speeded up those of deactivation, and shifted the opening kinetics by approximately + 60 mV. Interestingly, all these parameters of channel gating were not affected equally by La3+. Furthermore, amplitudes of the inward tail currents evoked at potentials more negative than the potassium equilibrium potential (E(K+)) were more strongly inhibited by La3+ than those of the outward tail currents evoked at potentials more positive than E(K+). This suggests voltage-dependent block and binding of La3+ to the Kv1.1 channel protein. We conclude that these actions cannot be explained in terms of surface charge considerations alone. Our results provide evidence for a direct interaction with the potassium channel protein, shedding new light on the mechanism of action of this lanthanide.

  8. Syntheses and biological activities of potent potassium channel openers derived from (+/-)-2-oxo-1-pyridin-3-yl-cyclohexanecarbothioic acid methylamide: new potassium channel openers.

    PubMed

    Brown, T J; Chapman, R F; Mason, J S; Palfreyman, M N; Vicker, N; Walsh, R J

    1993-05-28

    The syntheses and biological activities of (+/-)-2-(cyanomethylene)-1-pyridin-3-ylcyclohexanecarbothioic++ + acid methylamide (6) and trans-(+/-)-2-(cyanomethyl)-1-pyridin-3-ylcyclohexanecarbothioic acid methylamide (14) derived from (+/-)-2-oxo-1-pyridin-3-ylcyclohexanecarbothioic acid methylamide (4) are reported. Compounds were tested for antagonism of potassium-induced contraction of de-endothelialized rat aorta. The effects of modification of 6 and 14 on in vitro K(+)-channel opening activity are presented. These new series of potassium channel openers so derived are best exemplified by (+/-)-2-[2-(phenylsulfanyl)ethylidene]-1-pyridin-3-ylcyclohexan ecarbothioic acid methylamide (13d, RP 66266) and trans-(+/-)-2-[2-[(phenylsulfonyl)amino]ethyl]-1-pyridin-3- ylcyclohexanecarbothioic acid methylamide (25a, RP 66784), which have IC90 values of 3 and 0.3 nM, respectively. The potency of the most active compounds indicates a possible interaction at an extra binding site. The compounds described herein are potential antihypertensive and antianginal agents.

  9. Involvement of WNK1-mediated potassium channels in the sexual dimorphism of blood pressure.

    PubMed

    Yu, Guofeng; Cheng, Mengting; Wang, Wei; Zhao, Rong; Liu, Zhen

    2017-04-01

    Potassium homeostasis plays an essential role in the control of blood pressure. It is unknown, however, whether potassium balance is involved in the gender-associated blood pressure differences. We therefore investigated the possible mechanism of sexual dimorphism in blood pressure regulation by measuring the blood pressure, plasma potassium, renal actions of potassium channels and upstream regulator in male and female mice. Here we found that female mice exhibited lower blood pressure and higher plasma K(+) level as compared to male littermates. Western blot analyses of mouse kidney extract revealed a significant decrease in renal outer medullary potassium (ROMK) channel expression, while large-conductance Ca(2+)-activated K(+) (BK) channel and Na-K-2Cl cotransporter (NKCC2) as well as the upstream regulator with-no-lysine kinase 1 (WNK1) enhanced in female mice under normal condition. Surprisingly, both dietary K(+) loading and K(+) depletion eliminated the differences in plasma K(+) and blood pressure between females and males, and the differences of renal K(+) channels and WNK1 also attenuated in both groups of mice. These findings indicated the existence of a close correlation between K(+) homeostasis and sex-associated blood pressure. Moreover, the differential regulation of ROMK, BK-α and NKCC2 between female and male mice, at least, were partly mediated via WNK1 pathway, which may contribute to the sexual dimorphism of plasma K(+) and blood pressure control. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. 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.

  11. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation.

    PubMed

    Moha Ou Maati, H; Borsotto, M; Chatelain, F; Widmann, C; Lazdunski, M; Heurteaux, C

    2012-09-01

    NeuroAid (MLC601 and MLC901), a Traditional Medicine used in China for patients after stroke has been reported in preclinical models of ischemia to induce neuroprotection and neuroplasticity. This work shows the effects of MLC901 on an in vitro model of oxygen glucose deprivation (OGD). MLC901 prevents neuronal death induced by 120 min OGD and decreases the exaggerated Ca²⁺ entry in mature cortical neurons exposed to 120 min OGD. The neuroprotective effect of MLC901 is associated with a large hyperpolarization of ∼20 mV which is antagonized by glibenclamide, the specific inhibitor of K(ATP) channels. In addition MLC901 strengthens the activation of K(ATP) channels. MLC901 has been directly shown to act as an activator of K(ATP) channels as potent as the classical K(ATP) channel opener. The capacity of MLC901 to produce a large hyperpolarization, particularly in neurons that have suffered from energy deprivation probably plays an important role in the neuroprotective effects of this traditional medicine that comes in addition to its previously demonstrated neuroregenerative properties. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. hERG potassium channel blockade by the HCN channel inhibitor bradycardic agent ivabradine.

    PubMed

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

    2015-04-24

    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. Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Ih ERG 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 Ih ERG 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. 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 Ih ERG 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. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  14. 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

  15. Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants.

    PubMed

    Xiong, Qiaojie; Sun, Haiyan; Li, Min

    2007-05-01

    KCNQ potassium channels are activated by changes in transmembrane voltage and play an important role in controlling electrical excitability. Human mutations of KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of channel activity and cause benign familial neonatal convulsions (BFNCs). Thus, small molecules capable of augmenting KCNQ currents are essential both for understanding the mechanism of channel activity and for developing therapeutics. We performed a high-throughput screen in search for agonistic compounds potentiating KCNQ potassium channels. Here we report identification of a new opener, zinc pyrithione (1), which activates both recombinant and native KCNQ M currents. Interactions with the channel protein cause an increase of single-channel open probability that could fully account for the overall conductance increase. Separate point mutations have been identified that either shift the concentration dependence or affect potentiation efficacy, thereby providing evidence for residues influencing ligand binding and downstream events. Furthermore, zinc pyrithione is capable of rescuing the mutant channels causal to BFNCs.

  16. Importance of lipid-pore loop interface for potassium channel structure and function.

    PubMed

    van der Cruijsen, Elwin A W; Nand, Deepak; Weingarth, Markus; Prokofyev, Alexander; Hornig, Sönke; Cukkemane, Abhishek Arun; Bonvin, Alexandre M J J; Becker, Stefan; Hulse, Raymond E; Perozo, Eduardo; Pongs, Olaf; Baldus, Marc

    2013-08-06

    Potassium (i.e., K(+)) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K(+) channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K(+) channel inactivation and exhibits a remarkable structural plasticity that correlates to K(+) channel inactivation. The transmembrane helix 1 unwinds when the K(+) channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K(+) ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K(+) channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K(+) channel pore.

  17. Glucose deprivation activates diversity of potassium channels in cultured rat hippocampal neurons.

    PubMed

    Velasco, Myrian; García, Esperanza; Onetti, Carlos G

    2006-05-01

    1. Glucose is one of the most important substrates for generating metabolic energy required for the maintenance of cellular functions. Glucose-mediated changes in neuronal firing pattern have been observed in the central nervous system of mammals. K(+) channels directly regulated by intracellular ATP have been postulated as a linkage between cellular energetic metabolism and excitability; the functional roles ascribed to these channels include glucose-sensing to regulate energy homeostasis and neuroprotection under energy depletion conditions. The hippocampus is highly sensitive to metabolic insults and is the brain region most sensitive to ischemic damage. Because the identity of metabolically regulated potassium channels present in hippocampal neurons is obscure, we decided to study the biophysical properties of glucose-sensitive potassium channels in hippocampal neurons. 2. The dependence of membrane potential and the sensitivity of potassium channels to glucose and ATP in rat hippocampal neurons were studied in cell-attached and excised inside-out membrane patches. 3. We found that under hypoglycemic conditions, at least three types of potassium channels were activated; their unitary conductance values were 37, 147, and 241 pS in symmetrical K(+), and they were sensitive to ATP. For K(+) channels with unitary conductance of 37 and 241, when the membrane potential was depolarized the longer closed time constant diminished and this produced an increase in the open-state probability; nevertheless, the 147-pS channels were not voltage-dependent. 4. We propose that neuronal glucose-sensitive K(+) channels in rat hippocampus include subtypes of ATP-sensitive channels with a potential role in neuroprotection during short-term or prolonged metabolic stress.

  18. The role of Kv3-type potassium channels in cerebellar physiology and behavior.

    PubMed

    Joho, Rolf H; Hurlock, Edward C

    2009-09-01

    Different subunits of the Kv3 subfamily of voltage-gated potassium (Kv) channels (Kv3.1-Kv3.4) are expressed in distinct neuronal subpopulations in the cerebellum. Behavioral phenotypes in Kv3-null mutant mice such as ataxia with prominent hypermetria and heightened alcohol sensitivity are characteristic of cerebellar dysfunction. Here, we review how the unique biophysical properties of Kv3-type potassium channels, fast activation and fast deactivation that enable cerebellar neurons to generate brief action potentials at high frequencies, affect firing patterns and influence cerebellum-mediated behavior.

  19. Expression and function of potassium channels in the human placental vasculature.

    PubMed

    Wareing, Mark; Bai, Xilian; Seghier, Fella; Turner, Claire M; Greenwood, Susan L; Baker, Philip N; Taggart, Michael J; Fyfe, Gregor K

    2006-08-01

    In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.

  20. 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

  1. Animal toxins acting on voltage-gated potassium channels.

    PubMed

    Mouhat, Stéphanie; Andreotti, Nicolas; Jouirou, Besma; Sabatier, Jean-Marc

    2008-01-01

    Animal venoms are rich natural sources of bioactive compounds, including peptide toxins acting on the various types of ion channels, i.e. K(+), Na(+), Cl(-) and Ca(2+). Among K+ channel-acting toxins, those selective for voltage-gated K(+) (Kv) channels are widely represented and have been isolated from the venoms of numerous animal species, such as scorpions, sea anemones, snakes, marine cone snails and spiders. The toxins characterized hitherto contain between 22 and 60 amino acid residues, and are cross-linked by two to four disulfide bridges. Depending on their types of fold, toxins can be classified in eight structural categories, which showed a combination of beta-strands, helices, or a mixture of both. The main architectural motifs thereof are referred to as alpha/beta scaffold and inhibitor cystine knot (ICK). A detailed analysis of toxin structures and pharmacological selectivities indicates that toxins exhibiting a similar type of fold can exert their action on several subtypes of Kv channels, whereas a particular Kv channel can be targeted by toxins that possess unrelated folds. Therefore, it appears that the ability of structurally divergent toxins to interact with a particular Kv channel relies onto a similar spatial distribution of amino acid residues that are key to the toxin-channel interaction (rather than the type of toxin fold). The diversity of Kv channel blockers and their therapeutic value in the potential treatment of a number of specific human diseases, especially autoimmune disorders, inflammatory neuropathies and cancer, are reviewed.

  2. 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

  3. Identification of Putative Potassium Channel Homologues in Pathogenic Protozoa

    PubMed Central

    Prole, David L.; Marrion, Neil V.

    2012-01-01

    K+ channels play a vital homeostatic role in cells and abnormal activity of these channels can dramatically alter cell function and survival, suggesting that they might be attractive drug targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, leishmaniasis, trypanosomiasis and dysentery that are responsible for millions of deaths each year worldwide. The genomes of many protozoan parasites have recently been sequenced, allowing rational design of targeted therapies. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of K+ channels. These protozoan K+ channel homologues represent novel targets for anti-parasitic drugs. Differences in the sequences and diversity of human and parasite proteins may allow pathogen-specific targeting of these K+ channel homologues. PMID:22363819

  4. Sodium metabisulfite modulation of potassium channels in pain-sensing dorsal root ganglion neurons.

    PubMed

    Nie, Aifang; Wei, Cailing; Meng, Ziqiang

    2009-12-01

    The effects of sodium metabisulfite (SMB), a general food preservative, on potassium currents in rat dorsal root ganglion (DRG) neurons were investigated using the whole-cell patch-clamp technique. SMB increased the amplitudes of both transient outward potassium currents and delayed rectifier potassium current in concentration- and voltage-dependent manner. The transient outward potassium currents (TOCs) include a fast inactivating (A-current or IA) current and a slow inactivating (D-current or ID) current. SMB majorly increased IA, and ID was little affected. SMB did not affect the activation process of transient outward currents (TOCs), but the inactivation curve of TOCs was shifted to more positive potentials. The inactivation time constants of TOCs were also increased by SMB. For delayed rectifier potassium current (IK), SMB shifted the activation curve to hyperpolarizing direction. SMB differently affected TOCs and IK, its effects major on A-type K+ channels, which play a role in adjusting pain sensitivity in response to peripheral redox conditions. SMB did not increase TOCs and IK when adding DTT in pipette solution. These results suggested that SMB might oxidize potassium channels, which relate to adjusting pain sensitivity in pain-sensing DRG neurons.

  5. Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels

    PubMed Central

    Tang, Xiang D.; Daggett, Heather; Hanner, Markus; Garcia, Maria L.; McManus, Owen B.; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2001-01-01

    Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism. PMID:11222629

  6. 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

  7. Block of a Ca(2+)-activated potassium channel by cocaine.

    PubMed

    Premkumar, L S

    2005-04-01

    The primary target for cocaine is believed to be monoamine transporters because of cocaine's high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K(+) channel in hippocampal neurons grown in culture (IC(50)=approximately 30 microM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca(2+)-activated K(+ )channels are 261+/-37 microM: (-1)s(-1) and 11451+/-1467 s(-1). The equilibrium dissociation constant (K(B)) for cocaine, determined from single-channel parameters, is 43 microM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca(2+)-dependent K(+) channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.

  8. 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

  9. 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

  10. Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation.

    PubMed

    Perry, Matthew D; Rajendran, Vazhaikkurichi M; MacLennan, Kenneth A; Sandle, Geoffrey I

    2016-11-01

    Rat proximal and distal colon are net K(+) secretory and net K(+) absorptive epithelia, respectively. Chronic dietary K(+) loading increases net K(+) secretion in the proximal colon and transforms net K(+) absorption to net K(+) secretion in the distal colon, but changes in apical K(+) channel expression are unclear. We evaluated expression/activity of apical K(+) (BK) channels in surface colonocytes in proximal and distal colon of control and K(+)-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K(+) secretory proximal colon (39% of patches) than in those from K(+)-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K(+) loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K(+) loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K(+) secretory proximal colon than in K(+) absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K(+) secretory response to dietary K(+) loading. Copyright © 2016 the American Physiological Society.

  11. Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation

    PubMed Central

    Perry, Matthew D.; Rajendran, Vazhaikkurichi M.; MacLennan, Kenneth A.

    2016-01-01

    Rat proximal and distal colon are net K+ secretory and net K+ absorptive epithelia, respectively. Chronic dietary K+ loading increases net K+ secretion in the proximal colon and transforms net K+ absorption to net K+ secretion in the distal colon, but changes in apical K+ channel expression are unclear. We evaluated expression/activity of apical K+ (BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+ secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+ loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+ loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K+ secretory proximal colon than in K+ absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+ secretory response to dietary K+ loading. PMID:27609768

  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. Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells

    PubMed Central

    Purcell, Erin K.; Liu, Liqian; Thomas, Paul V.; Duncan, R. Keith

    2011-01-01

    The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitabilityare unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassiumcurrent by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (∼30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology. PMID:22046269

  14. Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

    PubMed

    Yuhas, W A; Fuchs, P A

    1999-11-01

    Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell.

  15. Novel Leishmania and Malaria Potassium Channels: Candidate Therapeutic Targets

    DTIC Science & Technology

    2005-08-01

    2-each for Plasmodium falciparum [PFK1 & PFK2], Toxoplasma gondii [TGK1 & TGK2], and 3 each for Leishmania major [LMK1, LMK2, & LMK3] and Trypanasoma...cloned from Leishmania major. • Two additional K+ channel genes identified and cloned from Toxoplasma gondii . * Three additional K+ channel genes...falciparum, T. gondii , and 3 each for L. major and T. cruzi). Using a combination of cultured mammalian cells and Xenopus oocytes for heterologous expression

  16. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation.

    PubMed

    Gründemann, Jan; Clark, Beverley A

    2015-09-22

    Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells.

  17. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

    PubMed Central

    Gründemann, Jan; Clark, Beverley A.

    2015-01-01

    Summary Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  18. Cross talk between activation and slow inactivation gates of Shaker potassium channels.

    PubMed

    Panyi, Gyorgy; Deutsch, Carol

    2006-11-01

    This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs(+) ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.

  19. Collateral response to activation of potassium channels in vivo.

    PubMed

    Lamping, K G

    1998-04-01

    Activation of ATP-sensitive K+ channels is involved in the coronary vascular response to decreases in perfusion pressure and ischemia. Since activation of ATP-sensitive K+ channels in collateral vessels may be important in determining flow to collateral-dependent myocardium, the ability of collaterals to respond to activation of the channel was tested. In the beating heart of dogs, we compared responses of non-collaterals less than 100 microns in diameter to collaterals of similar size using computer-controlled stroboscopic epi-illumination of the left ventricle coupled to a microscope-video system. Aprikalim, a selective activator of ATP-sensitive K+ channels (0.1-10 microM) produced similar dose-dependent dilation of non-collaterals and collaterals. Relaxation was decreased by inhibition of ATP-sensitive K+ channels with glibenclamide, but not by inhibition of nitric oxide synthase with nitro-L-arginine. Bradykinin (10-100 microM) produced similar dilation of non-collaterals and collaterals which was decreased by nitro-L-arginine but not glibenclamide. Thus, in microvascular collaterals, relaxation to both nitric oxide and activation of ATP-sensitive K+ channels is similar to non-collaterals.

  20. Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts.

    PubMed

    Sandle, G I; Warhurst, G; Butterfield, I; Higgs, N B; Lomax, R B

    1999-11-01

    Somatostatin is a powerful inhibitor of intestinal Cl(-) secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K(+) channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl(-) secretory process. Somatostatin (2 microM) elicited a >80% decrease in "spontaneous" K(+) channel activity in cell-attached patches in nonstimulated crypts (50% inhibition = approximately 8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100-200 microM dibutyryl cAMP, 2 microM somatostatin and its synthetic analog octreotide (2 microM) both produced similar degrees of K(+) channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl(-) (5 mM) conditions. In addition, 2 microM somatostatin abolished the increase in K(+) channel activity stimulated by 2 microM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca(2+). These results indicate that somatostatin peptides inhibit 23-pS basolateral K(+) channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca(2+) sensitivity.

  1. Inward Rectifier Potassium Channels Control Rotor Frequency in Ventricular Fibrillation

    PubMed Central

    Jalife, José

    2009-01-01

    Summary Ventricular fibrillation (VF) is the most important cause of sudden cardiac death. While traditionally thought to result from random activation of the ventricles by multiple independent wavelets, recent evidence suggests that VF may be determined by the sustained activation of a relatively small number of reentrant sources. In addition, recent experimental data in various species as well as computer simulations have provided important clues about its ionic and molecular mechanisms, particularly in regards to the role of potassium currents in such mechanisms. The results strongly argue that the inward rectifier current, Ik1, is an important current during functional reentry because it mediates the electrotonic interactions between the unexcited core and its immediate surroundings. In addition, IK1 is a stabilizer of reentry due to its ability to shorten action potential duration and reducing conduction velocity near the center of rotation. Increased I K1 prevents wavefront-wavetail interactions and thus averts rotor destabilization and breakup. Other studies have shown that while the slow component of the delayed rectifier potassium current, IKs, does not significantly modify rotor frequency or stability, it plays a major role in post-repolarization refractoriness and wavebreak formation. Therefore, the interplay between IK1 and the rapid sodium inward current (INa) is a major factor in the control of cardiac excitability and therefore the stability and frequency of reentry while IKs is an important determinant of fibrillatory conduction. PMID:19880073

  2. Contribution of potassium channels to the discharge properties of rat aortic baroreceptor sensory endings.

    PubMed

    Reynolds, P J; Yang, M; Andresen, M C

    1994-11-28

    The expression of several types of membrane potassium channel at the cell body and central synaptic terminal of the rat aortic arch baroreceptor has been reported by others. It is not known if any of the same channels function at the peripheral sensory terminal of these afferent nerves. Our study examined the effect of three potassium channel blocking agents on the pressure-evoked discharge of such baroreceptors. Thirty-one single unit, regularly discharging baroreceptors were studied using an in vitro aortic arch-aortic nerve preparation. Discharge thresholds and suprathreshold pressure sensitivities were derived from responses of receptors to slowly rising ramps of pressure applied to the aortic arch. Vessel diameter was recorded along with receptor discharge to assess any drug-induced changes in vascular smooth muscle. The blocking agents tested have a range of specificities for classes of potassium channels: tetraethylammonium (TEA), 4-aminopyridine (4-AP) and charybdotoxin. TEA depressed the pressure sensitivity of all baroreceptors tested (n = 3) in a dose-dependent manner. Baroreceptor responses to 4-AP were complex (n = 22) and varied widely across individuals. Three were unaffected by 5 mM 4-AP. Most baroreceptors were generally depressed by 4-AP. Some of the 4-AP effects appeared to be related to actions at vascular smooth muscle. None of the baroreceptors tested (n = 6) was affected by charybdotoxin. The results of selective potassium channel blockade are generally consistent with what would be expected from a sustained depolarization of baroreceptor endings such as has been reported with raising extracellular potassium and probably includes effects of inactivation of other voltage-dependent channels.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack

    PubMed Central

    Brown, Maile R.; Kronengold, Jack; Gazula, Valeswara-Rao; Chen, Yi; Strumbos, John G.; Sigworth, Fred J.; Navaratnam, Dhasakumar; Kaczmarek, Leonard K.

    2010-01-01

    In humans, absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein, results in Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Here we report through biochemical and electrophysiological studies that FMRP binds the C-terminus of the Slack sodium-activated potassium channel to activate the channel. The findings suggest that Slack activity may provide a link between patterns of neuronal firing and changes in protein translation. PMID:20512134

  4. Escitalopram block of hERG potassium channels.

    PubMed

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

    2014-01-01

    Escitalopram, a selective serotonin reuptake inhibitor, is the pharmacologically active S-enantiomer of the racemic mixture of RS-citalopram and is widely used in the treatment of depression. The effects of escitalopram and citalopram on the human ether-a-go-go-related gene (hERG) channels expressed in human embryonic kidney cells were investigated using voltage-clamp and Western blot analyses. Both drugs blocked hERG currents in a concentration-dependent manner with an IC50 value of 2.6 μM for escitalopram and an IC50 value of 3.2 μM for citalopram. The blocking of hERG by escitalopram was voltage-dependent, with a steep increase across the voltage range of channel activation. However, voltage independence was observed over the full range of activation. The blocking by escitalopram was frequency dependent. A rapid application of escitalopram induced a rapid and reversible blocking of the tail current of hERG. The extent of the blocking by escitalopram during the depolarizing pulse was less than that during the repolarizing pulse, suggesting that escitalopram has a high affinity for the open state of the hERG channel, with a relatively lower affinity for the inactivated state. Both escitalopram and citalopram produced a reduction of hERG channel protein trafficking to the plasma membrane but did not affect the short-term internalization of the hERG channel. These results suggest that escitalopram blocked hERG currents at a supratherapeutic concentration and that it did so by preferentially binding to both the open and the inactivated states of the channels and by inhibiting the trafficking of hERG channel protein to the plasma membrane.

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

    PubMed

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

    1997-06-15

    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.

  6. Coordinated signal integration at the M-type potassium channel upon muscarinic stimulation.

    PubMed

    Kosenko, Anastasia; Kang, Seungwoo; Smith, Ida M; Greene, Derek L; Langeberg, Lorene K; Scott, John D; Hoshi, Naoto

    2012-05-29

    Several neurotransmitters, including acetylcholine, regulate neuronal tone by suppressing a non-inactivating low-threshold voltage-gated potassium current generated by the M-channel. Agonist dependent control of the M-channel is mediated by calmodulin, activation of anchored protein kinase C (PKC), and depletion of the phospholipid messenger phosphatidylinositol 4,5-bisphosphate (PIP2). In this report, we show how this trio of second messenger responsive events acts synergistically and in a stepwise manner to suppress activity of the M-current. PKC phosphorylation of the KCNQ2 channel subunit induces dissociation of calmodulin from the M-channel complex. The calmodulin-deficient channel has a reduced affinity towards PIP2. This pathway enhances the effect of concomitant reduction of PIP2, which leads to disruption of the M-channel function. These findings clarify how a common lipid cofactor, such as PIP2, can selectively regulate ion channels.

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

    PubMed Central

    Diness, Jonas G.; Bentzen, Bo H.; Sørensen, Ulrik S.

    2015-01-01

    Abstract: Small-conductance Ca2+-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

  8. Effectiveness of copper sulfate and potassium permanganate on channel catfish infected with Flavobacterium columnare

    USDA-ARS?s Scientific Manuscript database

    Copper sulfate (CuSO4) and potassium permanganate (KMnO4) were evaluated for their effectiveness to curtail mortality and decrease bacterial load in fish tissues and water in channel catfish Ictalurus punctatus naturally infected with Flavobacterium columnare, the causative agent of columnaris. Fis...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. Disease mechanisms in MS: the potassium channel KIR4.1--a potential autoantigen in MS.

    PubMed

    Racke, Michael K

    2012-11-05

    Multiple sclerosis is an inflammatory, demyelinating disease in which antigens of the myelin sheath have been considered the autoimmune target. A recent study suggests that the potassium channel KIR4.1 is another potential autoantigen in some patients with multiple sclerosis, and might also be a target in other demyelinating diseases.

  11. 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

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

    PubMed

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

    2016-06-01

    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. 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. 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. 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.

  13. 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.

  14. Differential distribution of the sodium-activated potassium channels slick and slack in mouse brain.

    PubMed

    Rizzi, Sandra; Knaus, Hans-Günther; Schwarzer, Christoph

    2016-07-01

    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.

  15. 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

  16. Voltage sensor ring in a native structure of a membrane-embedded potassium channel

    PubMed Central

    Shi, Liang; Zheng, Hongjin; Zheng, Hui; Borkowski, Brian A.; Shi, Dan; Gonen, Tamir; Jiang, Qiu-Xing

    2013-01-01

    Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid–protein interactions in general and for the mechanism of voltage sensing in particular. PMID:23401554

  17. Protective Roles for Potassium SK/KCa2 Channels in Microglia and Neurons

    PubMed Central

    Dolga, Amalia M.; Culmsee, Carsten

    2012-01-01

    New concepts on potassium channel function in neuroinflammation suggest that they regulate mechanisms of microglial activation, including intracellular calcium homeostasis, morphological alterations, pro-inflammatory cytokine release, antigen presentation, and phagocytosis. Although little is known about voltage independent potassium channels in microglia, special attention emerges on small (SK/KCNN1-3/KCa2) and intermediate (IK/KCNN4/KCa3.1)-conductance calcium-activated potassium channels as regulators of microglial activation in the field of research on neuroinflammation and neurodegeneration. In particular, recent findings suggested that SK/KCa2 channels, by regulating calcium homeostasis, may elicit a dual mechanism of action with protective properties in neurons and inhibition of inflammatory responses in microglia. Thus, modulating SK/KCa2 channels and calcium signaling may provide novel therapeutic strategies in neurological disorders, where neuronal cell death and inflammatory responses concomitantly contribute to disease progression. Here, we review the particular role of SK/KCa2 channels for [Ca2+]i regulation in microglia and neurons, and we discuss the potential impact for further experimental approaches addressing novel therapeutic strategies in neurological diseases, where neuronal cell death and neuroinflammatory processes are prominent. PMID:23189056

  18. Single potassium channels opened by opioids in rat locus ceruleus neurons.

    PubMed

    Miyake, M; Christie, M J; North, R A

    1989-05-01

    Currents through single-ion channels were recorded in the cell-attached configuration from locus ceruleus neurons enzymatically dissociated from newborn rats. When the selective mu opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol was in the patch-clamp electrode, unitary inward currents were observed with conductance of approximately 45 pS (measured at zero pipette potential, with 150 mM potassium in the recording electrode). Long silences, lasting many seconds to minutes, separated periods of activity of similar durations. Within such activity periods the distribution of closed times of the channels was best fitted by the sum of two exponential functions (time constants approximately 1 and 30 ms), and the durations of channel openings were fit by a single exponential function; mean open time increased from 2 to 120 ms as agonist concentration increased. Channel activity was not seen when high concentrations of opioids were applied to the neuron outside the patch-clamp recording electrode, indicating intimate coupling between receptor and potassium channel. Unitary currents with similar properties were also seen when pipettes contained alpha 2 adrenoceptor agonists or somatostatin. Taken with previous findings, the results indicate that mu opioid receptors, alpha 2 adrenoceptors, and somatostatin receptors can couple directly to membrane potassium channels through the local intermediary action of a GTP binding protein.

  19. 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

  20. Initial steps in the opening of a Shaker potassium channel.

    PubMed

    Hoshi, Toshinori; Armstrong, Clay M

    2012-07-31

    The structural model of a K(V) (K(+)-selective, voltage-gated) channel in the open state is known (Protein Data Bank ID code 2R9R). Each subunit of the channel has four negatively charged residues distributed in the transmembrane segments S1, S2, and S3 that bind to and facilitate the movement within the membrane of the positively charged, voltage-sensing residues of S4. When extrapolated to the closed state, the two outermost negatively charged residues are exposed to extracellular fluid and not bound to S4 residues, all of which have theoretically been driven inward by voltage. If this closed state model is correct, these residues are available to bind external cations. We examined the effects of La(3+) on voltage-gated Shaker K(+) channels. Addition of the trivalent cation La(3+) (50 μM) extracellularly markedly prolongs the lag that precedes channel opening and slows the subsequent rise of K(+) current (I(K)) at all voltages. Decay kinetics of I(K) at negative voltages are unaltered. Gating current (I(g)) recorded from a nonconducting mutant shows that La(3+) reduces the initial amplitude of I(g) nearly twofold. We postulate that, in the resting state, La(3+) binds to the unoccupied, outermost negative residues, hindering outward S4 motion, thus increasing the lag on activation and slowing the rise of I(K). In the activated state, La(3+) is displaced by outward movement of arginine residues in S4; La(3+), therefore, is not present to affect channel closing. The results give strong support to the closed state model of the K(V) channel and a clear explanation of the effect of multivalent cations on cellular excitability.

  1. Molecular Diversity and Functional Evolution of Scorpion Potassium Channel Toxins*

    PubMed Central

    Zhu, Shunyi; Peigneur, Steve; Gao, Bin; Luo, Lan; Jin, Di; Zhao, Yong; Tytgat, Jan

    2011-01-01

    Scorpion toxins affecting K+ channels (KTxs) represent important pharmacological tools and potential drug candidates. Here, we report molecular characterization of seven new KTxs in the scorpion Mesobuthus eupeus by cDNA cloning combined with biochemical approaches. Comparative modeling supports that all these KTxs share a conserved cysteine-stabilized α-helix/β-sheet structural motif despite the differences in protein sequence and size. We investigated functional diversification of two orthologous α-KTxs (MeuTXKα1 from M. eupeus and BmP01 from Mesobuthus martensii) by comparing their K+ channel-blocking activities. Pharmacologically, MeuTXKα1 selectively blocked Kv1.3 channel with nanomolar affinity (IC50, 2.36 ± 0.9 nm), whereas only 35% of Kv1.1 currents were inhibited at 3 μm concentration, showing more than 1271-fold selectivity for Kv1.3 over Kv1.1. This peptide displayed a weak effect on Drosophila Shaker channel and no activity on Kv1.2, Kv1.4, Kv1.5, Kv1.6, and human ether-a-go-go-related gene (hERG) K+ channels. Although BmB01 and MeuTXKα1 have a similar channel spectrum, their affinity and selectivity for these channels largely varies. In comparison with MeuTXKα1, BmP01 only exhibits a submicromolar affinity (IC50, 133.72 ± 10.98 nm) for Kv1.3, showing 57-fold less activity than MeuTXKα1. Moreover, it lacks the ability to distinguish between Kv1.1 and Kv1.3. We also found that MeuTXKα1 inhibited the proliferation of activated T cells induced by phorbol myristate acetate and ionomycin at micromolar concentrations. Our results demonstrate that accelerated evolution drives affinity variations of orthologous α-KTxs on Kv channels and indicate that MeuTXKα1 is a promising candidate to develop an immune modulation agent for human autoimmune diseases. PMID:20889474

  2. Oxygen-induced tension in the sheep ductus arteriosus: effects of gestation on potassium and calcium channel regulation

    PubMed Central

    Waleh, Nahid; Reese, Jeff; Kajino, Hiroki; Roman, Christine; Seidner, Steven; McCurnin, Donald; Clyman, Ronald I.

    2009-01-01

    Compared with the full term ductus arteriosus, the premature ductus is less likely to constrict when exposed to postnatal oxygen concentrations. We used isolated fetal sheep ductus arteriosus (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine if changes in K+- and CaL-channel activity could account for the developmental differences in oxygen-induced tension. In the mature ductus, KV-channels appear to be the only K+-channels that oppose ductus tension. Oxygen concentrations between (2 and 15%) inhibit KV-channel activity, which increases the CaL-channel-mediated increase in tension. Low oxygen concentrations have a direct inhibitory effect on CaL-channel activity in the immature ductus; this is not the case in the mature ductus. In the immature ductus, 3 different K+-channel activities (KV, KCa, and KATP) oppose ductus tension and contribute to its decreased tone. Oxygen inhibits the activities of all 3 K+-channels. The inhibitory effects of the 3 K+-channel activities decline with advancing gestation. The decline in K+-channel activity is not due to decreased K+-channel expression. Super-physiologic oxygen concentrations (≥30% O2) constrict the ductus by utilizing calcium dependent pathways that are independent of K+- and CaL-channel activities. Super-physiologic oxygen concentrations eliminate the difference in tensions between the 2 age groups. PMID:19092721

  3. Resveratrol attenuates cortical neuron activity: roles of large conductance calcium-activated potassium channels and voltage-gated sodium channels.

    PubMed

    Wang, Ya-Jean; Chan, Ming-Huan; Chen, Linyi; Wu, Sheng-Nan; Chen, Hwei-Hisen

    2016-05-21

    Resveratrol, a phytoalexin found in grapes and red wine, exhibits diverse pharmacological activities. However, relatively little is known about whether resveratrol modulates the ion channels in cortical neurons. The large-conductance calcium-activated potassium channels (BKCa) and voltage-gated sodium channels were expressed in cortical neurons and play important roles in regulation of neuronal excitability. The present study aimed to determine the effects of resveratrol on BKCa currents and voltage-gated sodium currents in cortical neurons. Resveratrol concentration-dependently increased the current amplitude and the opening activity of BKCa channels, but suppressed the amplitude of voltage-gated sodium currents. Similar to the BKCa channel opener NS1619, resveratrol decreased the firing rate of action potentials. In addition, the enhancing effects of BKCa channel blockers tetraethylammonium (TEA) and paxilline on action potential firing were sensitive to resveratrol. Our results indicated that the attenuation of action potential firing rate by resveratrol might be mediated through opening the BKCa channels and closing the voltage-gated sodium channels. As BKCa channels and sodium channels are critical molecular determinants for seizure generation, our findings suggest that regulation of these two channels in cortical neurons probably makes a considerable contribution to the antiseizure activity of resveratrol.

  4. Kv3 voltage-gated potassium channels regulate neurotransmitter release from mouse motor nerve terminals.

    PubMed

    Brooke, Ruth E; Moores, Thomas S; Morris, Neil P; Parson, Simon H; Deuchars, Jim

    2004-12-01

    Voltage-gated potassium (Kv) channels are critical to regulation of neurotransmitter release throughout the nervous system but the roles and identity of the subtypes involved remain unclear. Here we show that Kv3 channels regulate transmitter release at the mouse neuromuscular junction (NMJ). Light- and electron-microscopic immunohistochemistry revealed Kv3.3 and Kv3.4 subunits within all motor nerve terminals of muscles examined [transversus abdominus, lumbrical and flexor digitorum brevis (FDB)]. To determine the roles of these Kv3 subunits, intracellular recordings were made of end-plate potentials (EPPs) in FDB muscle fibres evoked by electrical stimulation of tibial nerve. Tetraethylammonium (TEA) applied at low concentrations (0.05-0.5 mM), which blocks only a few known potassium channels including Kv3 channels, did not affect muscle fibre resting potential but significantly increased the amplitude of all EPPs tested. Significantly, this effect of TEA was still observed in the presence of the large-conductance calcium-activated potassium channel blockers iberiotoxin (25-150 nM) and Penitrem A (100 nM), suggesting a selective action on Kv3 subunits. Consistent with this, 15-microM 4-aminopyridine, which blocks Kv3 but not large-conductance calcium-activated potassium channels, enhanced evoked EPP amplitude. Unexpectedly, blood-depressing substance-I, a toxin selective for Kv3.4 subunits, had no effect at 0.05-1 microM. The combined presynaptic localization of Kv3 subunits and pharmacological enhancement of EPP amplitude indicate that Kv3 channels regulate neurotransmitter release from presynaptic terminals at the NMJ.

  5. 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.

  6. Schistosoma mansoni infection enhances host portal vein contraction: role of potassium channels and p38 MAP kinase.

    PubMed

    Araujo, F P; Quintas, L E M; Noël, F; Silva, C L M

    2007-07-01

    Murine Schistosoma mansoni infection is related to an increased contraction of portal vein in response to 5-hydroxytryptamine (5-HT). The present study addressed a putative alteration of ion channels and enzymes involved in vascular contraction. In control group, either inhibition of K+ channels sensitive to ATP (K(ATP)) or Ca2+ (BK(Ca)) increased 5-HT-induced contraction, but the same did not occur in infected mice. On the other hand, inhibition of p38 MAP kinase markedly decreased the vascular contraction to 5-HT in the infected mice with minor effects in the control group. Accordingly, we observed a higher density of phospho-p38 MAP kinase, that refers to the fully active state of the enzyme, in portal veins from infected mice as compared to control animals. These results suggest that the reduced function of K(ATP) and BK(Ca) channels along with an increased contribution of p38 MAP kinase contribute to the increased contraction of portal veins to 5-HT observed in murine schistosomiasis.

  7. 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.

  8. Athermalized channeled spectropolarimetry using a biaxial potassium titanyl phosphate crystal.

    PubMed

    Craven-Jones, Julia; Way, Brandyn M; Kudenov, Michael W; Mercier, Jeffrey A

    2013-05-15

    Channeled spectropolarimeters measure the polarization state of light as a function of wavelength. Typically, a channeled spectropolarimeter uses high-order retarders made of uniaxial crystal to amplitude modulate the measured spectrum with the Stokes polarization information. A primary limitation of these instruments is the thermal variability of the retarders, which necessitates frequent system recalibration. Past work has addressed this issue by implementing an athermalized retarder produced from two uniaxial crystals. However, reducing the complexity of an athermalized retarder is advantageous for minimizing size and weight requirements. In this Letter, a technique for producing a thermally stable channeled spectropolarimeter using biaxial retarders is presented. This technique preserves a constant phase over an appreciable temperature range. Proof-of-concept results from a KTP-based athermal partial channeled spectropolarimeter are presented from 500 to 750 nm for temperature changes up to 26°C. Spectropolarimetric reconstructions produced from this system vary by <=2.6% RMS when the retarder experiences a 13°C increase in temperature above 21°C ambient, <=5.2% for a 20°C increase, and <=6.7% for a 26°C increase.

  9. 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

  10. Nicotine depresses the functions of multiple cardiac potassium channels.

    PubMed

    Wang, H; Shi, H; Wang, Z

    1999-01-01

    Nicotine is the main constituent of tobacco smoke responsible for the elevated risk of the cardiovascular disease and sudden coronary death associated with smoking, presumably by provoking cardiac arrhythmias. The cellular mechanisms may be related to the ability of nicotine to prolong action potentials and to depolarize membrane potential. However, the underlying ionic mechanisms remained unknown. We showed here that nicotine blocked multiple types of K+ currents, including the native currents in canine ventricular myocytes and the cloned channels expressed in Xenopus oocytes: A-type K+ currents (I(to)/Kv4.3), delayed rectifier K+ currents (I(Kr)/HERG) and inward rectifier K+ currents (I(K1)/Kir2.1). Most noticeably, nicotine at a concentration as low as of 10 nM significantly suppressed I(to) and Kv4.3 by approximately 20%. The effects of nicotine were independent of nicotinic receptor simulation or catecholamine release. Our results indicate that nicotine is a non-specific blocker of K+ channels and the inhibitory effects are the consequence of direct interactions between nicotine molecules and the channel proteins. Our study provided for the first time the evidence for the direct inhibition of cardiac K+ channels by nicotine and established a novel aspect of nicotine pharmacology.

  11. Atomic basis for therapeutic activation of neuronal potassium channels.

    PubMed

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

    2015-09-03

    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.

  12. State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy.

    PubMed

    Ganapathi, Sindura B; Kester, Mark; Elmslie, Keith S

    2009-04-01

    Human ether-a-go-go-related gene (HERG) potassium channel acts as a delayed rectifier in cardiac myocytes and is an important target for both pro- and antiarrhythmic drugs. Many drugs have been pulled from the market for unintended HERG block causing arrhythmias. Conversely, recent evidence has shown that HERG plays a role in cell proliferation and is overexpressed both in multiple tumor cell lines and in primary tumor cells, which makes HERG an attractive target for cancer treatment. Therefore, a drug that can block HERG but that does not induce cardiac arrhythmias would have great therapeutic potential. Roscovitine is a cyclin-dependent kinase (CDK) inhibitor that is in phase II clinical trials as an anticancer agent. In the present study we show that R-roscovitine blocks HERG potassium current (human embryonic kidney-293 cells stably expressing HERG) at clinically relevant concentrations. The block (IC(50) = 27 microM) was rapid (tau = 20 ms) and reversible (tau = 25 ms) and increased with channel activation, which supports an open channel mechanism. Kinetic study of wild-type and inactivation mutant HERG channels supported block of activated channels by roscovitine with relatively little effect on either closed or inactivated channels. A HERG gating model reproduced all roscovitine effects. Our model of open channel block by roscovitine may offer an explanation of the lack of arrhythmias in clinical trials using roscovitine, which suggests the utility of a dual CDK/HERG channel block as an adjuvant cancer therapy.

  13. Control of anterior pituitary cell excitability by calcium-activated potassium channels.

    PubMed

    Shipston, Michael J

    2017-06-05

    In anterior pituitary endocrine cells, large (BK), small (SK) and intermediate (IK) conductance calcium activated potassium channels are key determinants in shaping cellular excitability in a cell type- and context-specific manner. Indeed, these channels are targeted by multiple signaling pathways that stimulate or inhibit cellular excitability. BK channels can, paradoxically, both promote electrical bursting as well as terminate bursting and spiking dependent upon intrinsic BK channel properties and proximity to voltage gated calcium channels in somatotrophs, lactotrophs and corticotrophs. In contrast, SK channels are predominantly activated by calcium released from intracellular IP3-sensitive calcium stores and mediate membrane hyperpolarization in cells including gonadotrophs and corticotrophs. IK channels are predominantly expressed in corticotrophs where they limit membrane excitability. A major challenge for the future is to determine the cell-type specific molecular composition of calcium-activated potassium channels and how they control anterior pituitary hormone secretion as well as other calcium-dependent processes. Copyright © 2017. Published by Elsevier B.V.

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

    PubMed

    Melgari, Dario; Du, Chunyun; El Harchi, Aziza; Zhang, Yihong; Hancox, Jules C

    2014-10-01

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

  15. Hypoglycemia-activated K+ channels in hippocampal neurons.

    PubMed

    Tromba, C; Salvaggio, A; Racagni, G; Volterra, A

    1992-08-31

    Channels linking the electrical and metabolic activities of cells (KATP channels) have been described in various tissues, including some brain areas (hypothalamus, cerebral cortex and substantia nigra). Here we report the existence in hippocampal neurons of K+ permeant channels whose activity is regulated by extracellular glucose. They are open at the cell resting potential and respond to transient hypoglycemia with a reversible increase in activity. The one type so far characterized has a conductance of approximately 100 pS in isotonic K+, is inhibited by the sulphonylurea glibenclamide (1 microM), and is activated by the potassium channel opener lemakalim (0.1-1 microM). These data provide a direct demonstration of the presence, in hippocampal neurons, of glucose-sensitive channels that could belong to the KATP family.

  16. In vivo Expression of a Light-activatable Potassium Channel Using Unnatural Amino Acids

    PubMed Central

    Kang, Ji-Yong; Kawaguchi, Daichi; Coin, Irene; Xiang, Zheng; O’Leary, Dennis D. M.; Slesinger, Paul A.; Wang, Lei

    2013-01-01

    SUMMARY Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been employed to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly-rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel non-conducting, and upon brief light illumination, was released to permit outward K+ current. Expression of this photo-inducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expressed PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. The principles applied here to a potassium channel could be generally expanded to other proteins expressed in the brain to enable optical regulation. PMID:24139041

  17. Characterization of potassium channel modulators with QPatch automated patch-clamp technology: system characteristics and performance.

    PubMed

    Kutchinsky, Jonatan; Friis, Søren; Asmild, Margit; Taboryski, Rafael; Pedersen, Simon; Vestergaard, Ras K; Jacobsen, Rasmus B; Krzywkowski, Karen; Schrøder, Rikke L; Ljungstrøm, Trine; Hélix, Nathalie; Sørensen, Claus B; Bech, Morten; Willumsen, Niels J

    2003-10-01

    Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

  18. Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

    PubMed Central

    Sánchez-Ponce, Diana; DeFelipe, Javier; Garrido, Juan José; Muñoz, Alberto

    2012-01-01

    Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation. PMID:23119056

  19. 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

  20. Artificial phosphorylation sites modulate the activity of a voltage-gated potassium channel

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Zocchi, Giovanni

    2015-03-01

    The KvAP potassium channel is representative of a family of voltage-gated ion channels where the membrane potential is sensed by a transmembrane helix containing several positively charged arginines. Previous work by Wang and Zocchi [A. Wang and G. Zocchi, PLoS ONE 6, e18598 (2011), 10.1371/journal.pone.0018598] showed how a negatively charged polyelectrolyte attached in proximity to the voltage sensing element can bias the opening probability of the channel. Here we introduce three phosphorylation sites at the same location and show that the response curve of the channel shifts by about 20 mV upon phosphorylation, while other characteristics such as the single-channel conductance are unaffected. In summary, we construct an artificial phosphorylation site which confers allosteric regulation to the channel.