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Sample records for potassium katp channel

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

    PubMed Central

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

    2016-01-01

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

  2. 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

  3. 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.

  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. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  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. 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.

  12. Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules.

    PubMed

    Yang, Shao-Nian; Wenna, Nancy Dekki; Yu, Jia; Yang, Guang; Qiu, Hua; Yu, Lina; Juntti-Berggren, Lisa; Köhler, Martin; Berggren, Per-Olof

    2007-09-01

    beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.

  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. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  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. 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

  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. 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.

  18. 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.

  19. 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

  20. 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...Diet and Potassium Channel Function 5b. GRANT NUMBER W81XWH-13-1-0463 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Geoffrey Murphy 5d. PROJECT...regulates neuronal excitability by influencing potassium channel activity via the auxiliary potassium channel subunit Kvβ2. To test this hypothesis we

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

    PubMed

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

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

  2. 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.

  3. 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.

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

    PubMed 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

  5. 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

  6. 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.

  7. Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

    PubMed Central

    Sierra, Ana; Subbotina, Ekaterina; Zhu, Zhiyong; Gao, Zhan; Koganti, Siva Rama Krishna; Coetzee, William; Goldhamer, David; Hodgson-Zingman, Denice M.; Zingman, Leonid V.

    2016-01-01

    Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities. PMID:26828268

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  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. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  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. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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."

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  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. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  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

    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.

  13. 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

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

    PubMed

    Dragicevic, E; Schiemann, J; Liss, B

    2015-01-22

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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. [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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

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

    PubMed

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

    2015-08-17

    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.

  17. 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

  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. 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

  20. 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.

  1. Activation or block of adenosine triphosphate-sensitive potassium channel have opposite effects on postcardioplegic myocardial dysfunction, "stunning". A multivariate prediction based on relative operating characteristic curve.

    PubMed

    Puddu, P E; Sugimoto, S; Monti, F; Iwashiro, K; Dawodu, A A; Schiariti, M; Chiavarelli, R; Marino, B; Campa, P P

    1995-09-01

    The relative effects of nicotinic acid (NA) and nitroglycerin (NT) added to cold high K+ cardioplegia were studied, to represent the two moieties of the adenosine triphosphate-sensitive potassium channel (KATP) activator nicorandil (N). In addition, we made a pooled analysis of a large series of experiments performed in our Laboratory to investigate the effects of KATP activation by N, or block (by glibenclamide, G), on postcardioplegic myocardial dysfunction. In both studies, reversibility from myocardial dysfunction (stunning) was assessed by the positive inotropic agent dobutamine. Guinea pig papillary muscle preparations were immersed in Tyrode's solution (O2 content 16 ml/l, 37 degrees C), then hypoxic (O2 content 5 ml/l) superfusion with hypothermic (20 degrees C) cardioplegic Saint Thomas' Hospital solution (STHS) was performed for 120 min. We investigated: A) 5 groups based on treatments added to STHS: 1) saline (Control (C)); 2) N = 1 mmol/L; 3) G = 1 mumol/L (also given for 15 min in Tyrode's solution); 4) NA = 1 mmol/L; 5) NT = 100 mumol/L; B) 76 consecutive experiments and we defined, independent of whether just before or during STHS: 1) KATP activation (by N, in the concentration range 1 mumol/L to 1 mmol/L, n = 36); 2) KATP block (by G 1 mumol/L, either alone or just before N, n = 20); 3) controls (n = 20) (either saline, n = 12, or saline plus dimethyl sulfoxide, as vehicle, at the ratio 100 to 1, n = 8). Absolute isometric contractility variables were evaluated along with percent changes of baseline values: 1) at 30 s of STHS, 2) after 60 min of reoxygenation with Tyrode's solution and 3) following further 15 min of dobutamine 10 mumol/L. In all preparations, developed tension (DT), time to peak tension (TPT), DT/TPT and time to arrest (TTA) were measured. In study A): TTA was significantly abbreviated (intergroup F = 5.79, p < 0.001) in N (49 +/- 11 s, mean +/- SD) p < 0.01 vs C and NA). At 30 s of STHS %DT/TPT was unchanged among groups. By

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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).

  17. 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

  18. 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.

  19. 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.

  20. 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).

  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. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

    2017-02-07

    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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

    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. PMID:27035963

  9. 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.

  10. 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…

  11. Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

    PubMed Central

    2013-01-01

    Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia. PMID:23764148

  12. 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.

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

    PubMed

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

    2014-10-01

    The Big Potassium (BK) ion channel is commonly known by a variety of names (Maxi-K, KCNMA1, slo, stretch-activated potassium channel, 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 the BK channel, especially its role, and its 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.

  14. 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).

  15. 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

  16. 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.

  17. 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.

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

    PubMed Central

    Momiyama, A; Takahashi, T

    1994-01-01

    The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission. PMID:7913967

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

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

    DTIC Science & Technology

    2005-08-01

    of Ca 2*-activated and voltage-gated K÷ channels) and verruclogen ( isolated from Penicillium verruculosum and a blocker of high conductance Ca 2...containing plasmid (plus 1/101h the amount of GFP voltage steps between -80 and 80mV. 0 = zero plasmid for identification ) by Lipofectamine 2000 current...assays were used to investigate parasite sensitivity to various K+ channel blocking compounds. The identification of only two putative K+ channels

  14. 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.

  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. 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.

  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. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  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. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. Atomic basis for therapeutic activation of neuronal potassium channels

    PubMed Central

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

    2015-01-01

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

  16. 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.

  17. 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

  18. 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.

  19. Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity.

    PubMed

    Ader, Christian; Schneider, Robert; Hornig, Sönke; Velisetty, Phanindra; Vardanyan, Vitya; Giller, Karin; Ohmert, Iris; Becker, Stefan; Pongs, Olaf; Baldus, Marc

    2009-09-16

    Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K(+) and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K(+) ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K(+)-channel pore.

  20. 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

  1. Biophysical Properties of ATP-sensitive Potassium Channels in CA3 Hippocampal Neurons

    NASA Astrophysics Data System (ADS)

    Obregón-Herrera, Armando; Márquez-Gamiño, Sergio; Onetti, Carlos G.

    2004-09-01

    Single-channel activity of glucose-sensitive channels from CA3 neurons of the rat hippocampus, was studied in cell-attached membrane patches. Single-channel activity was totally abolished at 20 mM external glucose. Glucose-sensitive channels were selective to K+ ions; the unitary conductance was 170 pS in 140 mM K+, and the K+ permeability was 3.86×10-13 cmṡs-1. The open-state probability (PO) increased with membrane depolarization as a result of mean open time enhancement and shortening of the closure periods. The activation midpoint was -79 mV. Glucose-sensitive K+ channel of CA3 neurons could be considered as an ATP-sensitive potassium channel.

  2. K2P Potassium Channels, Mysterious and Paradoxically Exciting

    PubMed Central

    Goldstein, Steve A. N.

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

  4. Model studies of the function of blockers on the small conductance potassium ion channel.

    PubMed

    Ciechanowicz-Rutkowska, M; Lewinski, K; Oleksyn, B; Stec, B

    2003-09-01

    A correlation between KI (equilibrium dissociation constants) and IC50 (concentration at 50% inhibition) inhibitors for the family of blockers of the small conductance potassium ion channels and their intrinsic characteristics like molecular mass and volume have been investigated. Most of the blockers in the family are not selective, in contrast to apamin - an 18 amino acid bee venom toxin - that is known to be a highly potent and selective blocker of these channels. Differences and similarities between the blockers have been analyzed, pointing toward the origin of their selectivity and relative potency. In conclusion, an ion channel blocking is a process controlled mainly by diffusion, in accordance with previous experimental results.

  5. Unitary conductance variation in Kir2.1 and in cardiac inward rectifier potassium channels.

    PubMed Central

    Picones, A; Keung, E; Timpe, L C

    2001-01-01

    Kir2.1 (IRK1) is the complementary DNA for a component of a cardiac inwardly rectifying potassium channel. When Kir2.1 is expressed in Xenopus oocytes or human embryonic kidney (HEK) cells (150 mM external KCl), the unitary conductances form a broad distribution, ranging from 2 to 33 pS. Channels with a similarly broad distribution of unitary conductance amplitudes are also observed in recordings from adult mouse cardiac myocytes under similar experimental conditions. In all three cell types channels with conductances smaller, and occasionally larger, than the ~30 pS ones are found in the same patches as the ~30 pS openings, or in patches by themselves. The unitary conductances in patches with a single active channel are stable for the durations of the recordings. Channels of all amplitudes share several biophysical characteristics, including inward rectification, voltage sensitivity of open probability, sensitivity of open probability to external divalent cations, shape of the open channel i-V relation, and Cs(+) block. The only biophysical difference found between large and small conductance channels is that the rate constant for Cs(+) block is reduced for the small-amplitude channels. The unblocking rate constant is similar for channels of different unitary conductances. Apparently there is significant channel-to-channel variation at a site in the outer pore or in the selectivity filter, leading to variability in the rate at which K(+) or Cs(+) enters the channel. PMID:11566776

  6. Unitary conductance variation in Kir2.1 and in cardiac inward rectifier potassium channels.

    PubMed

    Picones, A; Keung, E; Timpe, L C

    2001-10-01

    Kir2.1 (IRK1) is the complementary DNA for a component of a cardiac inwardly rectifying potassium channel. When Kir2.1 is expressed in Xenopus oocytes or human embryonic kidney (HEK) cells (150 mM external KCl), the unitary conductances form a broad distribution, ranging from 2 to 33 pS. Channels with a similarly broad distribution of unitary conductance amplitudes are also observed in recordings from adult mouse cardiac myocytes under similar experimental conditions. In all three cell types channels with conductances smaller, and occasionally larger, than the ~30 pS ones are found in the same patches as the ~30 pS openings, or in patches by themselves. The unitary conductances in patches with a single active channel are stable for the durations of the recordings. Channels of all amplitudes share several biophysical characteristics, including inward rectification, voltage sensitivity of open probability, sensitivity of open probability to external divalent cations, shape of the open channel i-V relation, and Cs(+) block. The only biophysical difference found between large and small conductance channels is that the rate constant for Cs(+) block is reduced for the small-amplitude channels. The unblocking rate constant is similar for channels of different unitary conductances. Apparently there is significant channel-to-channel variation at a site in the outer pore or in the selectivity filter, leading to variability in the rate at which K(+) or Cs(+) enters the channel.

  7. The cooperative voltage sensor motion that gates a potassium channel.

    PubMed

    Pathak, Medha; Kurtz, Lisa; Tombola, Francesco; Isacoff, Ehud

    2005-01-01

    The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.

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

    PubMed

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

    2016-06-01

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

  9. In Vivo Role of a Potassium Channel-binding Protein in Regulating Neuronal Excitability and Behavior

    PubMed Central

    Shahidullah, Mohammad; Reddy, Smitha; Fei, Hong; Levitan, Irwin B.

    2009-01-01

    Molecular details of ion channel interactions with modulatory subunits have been investigated widely in transfected cells, but the physiological roles of ion channel modulatory protein complexes in native neurons remain largely unexplored. The Drosophila large conductance calcium-activated potassium channel (dSlo) binds to and is modulated by its binding partner Slob. We have constructed flies in which Slob expression is manipulated by P-element mutagenesis, or by transgenic expression of Slob protein or Slob-RNAi. In vivo recordings of both macroscopic and single dSlo channel currents in identified neurosecretory neurons in the pars intercerebralis (PI) region of the Drosophila brain reveal that whole cell potassium current and properties of single dSlo channels are modulated by Slob expression level. Furthermore, Slob genotype influences action potential duration in vivo. This unprecedented combination of current clamp, macroscopic current and single channel recordings from neurons in brains of living flies defines a critical role for an ion channel modulatory protein complex in the control of neuronal excitability. We show further that Slob null flies exhibit significantly longer lifespan than controls under conditions of complete food deprivation. Crosses with deficiency lines demonstrate that this enhanced resistance to starvation-induced death maps close to the slob locus. Taken together, these results indicate that Slob may serve a novel regulatory function in feeding behavior, possibly by influencing the excitability of the PI neurons. PMID:19846720

  10. Activation of protein kinase C inhibits calcium-activated potassium channels in rat pituitary tumour cells.

    PubMed Central

    Shipston, M J; Armstrong, D L

    1996-01-01

    1. The regulation of large-conductance, calcium- and voltage-dependent potassium (BK) channels by protein kinase C (PKC) was investigated in clonal rat anterior pituitary cells (GH4C1), which were voltage clamped at -40 mV in a physiological potassium gradient through amphotericin-perforated patches. 2. Maximal activation of PKC by 100 nM phorbol 12, 13-dibutyrate (PdBu) almost completely inhibited the voltage-activated outward current through BK channels. In contrast PdBu had no significant effect on the residual outward current after block of BK channels with 2 mM TEA or 30 nM charybdotoxin. In single-channel recordings from cell-attached patches, PdBu reduced the open probability of BK channels more than eightfold with no significant effect on mean open lifetime or unitary conductance. 3. The effects of PdBu on BK channels were not mimicked by the 4 alpha-isomer, which does not activate PKC, and were blocked almost completely by 25 microM chelerythrine, a specific, noncompetitive PKC inhibitor. 4. PdBu had no significant effect on the amplitude of the pharmacologically isolated, high voltage-activated calcium current. 5. Inhibition of BK channel activity by PKC provides the first molecular mechanism linking hormonal activation of phospholipase C to sustained excitability in pituitary cells. PMID:8799890

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

    PubMed Central

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

    2012-01-01

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

  12. Inhibition of Kv4.3 potassium channels by trazodone.

    PubMed

    Chae, Yun Ju; Choi, Jin-Sung; Hahn, Sang June

    2013-08-01

    Trazodone, a triazolopyridine antidepressant, is commonly used in the treatment of depression and insomnia. Kv4.3 channels are transiently, and rapidly, inactivating Kv channels that are highly expressed in cardiac myocytes and neurons. To determine the electrophysiological basis for the cardiac and neuronal actions of trazodone, we studied the effects of trazodone on Kv4.3 currents stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Trazodone decreased the peak amplitude of Kv4.3 in a concentration-dependent manner with an IC50 of 55.4 μM. Under control conditions, the time course of inactivation of Kv4.3 at +40 mV was fitted to a double exponential function. Trazodone produced a concentration-dependent slowing of the fast and slow components of Kv4.3 inactivation during a voltage step to +40 mV. The inhibition of Kv4.3 by trazodone was voltage independent over the entire voltage range tested. Trazodone shifted the voltage dependence of the steady-state inactivation of Kv4.3 to a hyperpolarizing direction. However, the slope factor of the steady-state inactivation was not affected by trazodone. Under control conditions, the closed-state inactivation of Kv4.3 was fitted to a single exponential function. Trazodone significantly accelerated the closed-state inactivation of Kv4.3. Trazodone produced a weak use-dependent inhibition of Kv4.3 at frequencies of 1 and 2 Hz. m-Chlorophenylpiperazine (m-CPP), a major metabolite of trazodone, inhibited Kv4.3 less potently than trazodone, with an IC50 of 118.6 μM. These results suggest that trazodone preferentially inhibited Kv4.3 by both binding to the closed state and accelerating the closed-state inactivation of the channel.

  13. Direct block of inward rectifier potassium channels by nicotine.

    PubMed

    Wang, H; Yang, B; Zhang, L; Xu, D; Wang, Z

    2000-04-01

    Nicotine has been shown to depolarize membrane potential and to lengthen action potential duration in isolated cardiac preparations. To investigate whether this is a consequence of direct interaction of nicotine with inward rectifier K(+) channels which are a key determinant of membrane potentials, we assessed the effects of nicotine on two cloned human inward rectifier K(+) channels, Kir2.1 and Kir2.2, expressed in Xenopus oocytes and the native inward rectifier K(+) current I(K1) in canine ventricular myocytes. Nicotine suppressed Kir2.1-expressed currents at varying potentials negative to -20 mV, with more pronounced effects on the outward current between -70 and -20 mV relative to the inward current at hyperpolarized potentials (below -70 mV). The inhibition was concentration dependent. For the outward currents recorded at -50 mV, the IC50 was 165 +/- 18 microM. Similar effects of nicotine were observed for Kir2.2. A more potent effect was seen with I(K1) in canine myocytes. Significant blockade ( approximately 60%) was found at a concentration as low as 0.5 microM and the IC50 was 4.0 +/- 0.4 microM. The effects in both oocytes and myocytes were partially reversible upon washout of nicotine. Antagonists of nicotinic receptors (mecamylamine, 100 microM), muscarinic receptors (atropine, 1 microM), and beta-adrenergic receptors (propranolol, 1 microM) all failed to restore the depressed currents, suggesting that nicotine acted directly on Kir channels, independent of catecholamine release. This property of nicotine may explain its membrane-depolarizing and action potential duration-prolonging effects in cardiac cells and may contribute in part to its ability to promote propensity for cardiac arrhythmias.

  14. Voltage-gated potassium channel KCNV2 (Kv8.2) contributes to epilepsy susceptibility

    PubMed Central

    Jorge, Benjamin S.; Campbell, Courtney M.; Miller, Alison R.; Rutter, Elizabeth D.; Gurnett, Christina A.; Vanoye, Carlos G.; George, Alfred L.; Kearney, Jennifer A.

    2011-01-01

    Mutations in voltage-gated ion channels are responsible for several types of epilepsy. Genetic epilepsies often exhibit variable severity in individuals with the same mutation, which may be due to variation in genetic modifiers. The Scn2aQ54 transgenic mouse model has a sodium channel mutation and exhibits epilepsy with strain-dependent severity. We previously mapped modifier loci that influence Scn2aQ54 phenotype severity and identified Kcnv2, encoding the voltage-gated potassium channel subunit Kv8.2, as a candidate modifier. In this study, we demonstrate a threefold increase in hippocampal Kcnv2 expression associated with more severe epilepsy. In vivo exacerbation of the phenotype by Kcnv2 transgenes supports its identification as an epilepsy modifier. The contribution of KCNV2 to human epilepsy susceptibility is supported by identification of two nonsynonymous variants in epilepsy patients that alter function of Kv2.1/Kv8.2 heterotetrameric potassium channels. Our results demonstrate that altered potassium subunit function influences epilepsy susceptibility and implicate Kcnv2 as an epilepsy gene. PMID:21402906

  15. Expression of the two pore domain potassium channel TREK-1 in human intervertebral disc cells.

    PubMed

    Sharma, Pankaj; Hughes, Stephen; El Haj, Alicia; Maffulli, Nicola

    2012-07-01

    Potassium channels play a major role in intracellular homeostasis and regulation of cell volume. Intervertebral disc cells respond to mechanical loading in a complex manner. Mechanical loading may play a role in disc degeneration. Lumbar intervertebral disc samples from 5 patients (average age: 47 years, range: 25-64 years) were used for this study, investigating cells from the nucleus pulposus and the annulus fibrosus duplicate samples to determine RNA expression and protein expression. Analysis of mRNA expression by RT-PCR demonstrated that TREK 1 was expressed by nucleus pulposus (n=5) and annulus fibrosus (n=5) cells. Currently, TREK-1 is the only potassium channel known to be activated by intracellular acidosis, and responds to mechanical and chemical stimuli. Whilst the precise role of potassium channels in cellular homeostasis remains to be determined, TREK-1 may be important to protect disc cells against ischaemic damage, and subsequent disc degeneration, and may also play a role in effecting mechanotransduction. Further research is required to fully elucidate the role of the TREK-1 ion channel in intervertebral disc cells.

  16. Alcohol modulation of G-protein-gated inwardly rectifying potassium channels: from binding to therapeutics

    PubMed Central

    Bodhinathan, Karthik; Slesinger, Paul A.

    2014-01-01

    Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction. PMID:24611054

  17. AMIGO-Kv2.1 Potassium Channel Complex Is Associated With Schizophrenia-Related Phenotypes

    PubMed Central

    Peltola, Marjaana A.; Kuja-Panula, Juha; Liuhanen, Johanna; Võikar, Vootele; Piepponen, Petteri; Hiekkalinna, Tero; Taira, Tomi; Lauri, Sari E.; Suvisaari, Jaana; Kulesskaya, Natalia; Paunio, Tiina; Rauvala, Heikki

    2016-01-01

    The enormous variability in electrical properties of neurons is largely affected by a multitude of potassium channel subunits. Kv2.1 is a widely expressed voltage-dependent potassium channel and an important regulator of neuronal excitability. The Kv2.1 auxiliary subunit AMIGO constitutes an integral part of the Kv2.1 channel complex in brain and regulates the activity of the channel. AMIGO and Kv2.1 localize to the distinct somatodendritic clusters at the neuronal plasma membrane. Here we have created and characterized a mouse line lacking the AMIGO gene. Absence of AMIGO clearly reduced the amount of the Kv2.1 channel protein in mouse brain and altered the electrophysiological properties of neurons. These changes were accompanied by behavioral and pharmacological abnormalities reminiscent of those identified in schizophrenia. Concomitantly, we have detected an association of a rare, population-specific polymorphism of KV2.1 (KCNB1) with human schizophrenia in a genetic isolate enriched with schizophrenia. Our study demonstrates the involvement of AMIGO-Kv2.1 channel complex in schizophrenia-related behavioral domains in mice and identifies KV2.1 (KCNB1) as a strong susceptibility gene for schizophrenia spectrum disorders in humans. PMID:26240432

  18. The thermosensitive potassium channel TREK-1 contributes to coolness-evoked responses of Grueneberg ganglion neurons.

    PubMed

    Stebe, Sabrina; Schellig, Katharina; Lesage, Florian; Breer, Heinz; Fleischer, Joerg

    2014-01-01

    Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Shah, Niyathi Hegde; Aizenman, Elias

    2013-01-01

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

  1. Voltage-dependent potassium channels in activated rat microglia.

    PubMed Central

    Nörenberg, W; Gebicke-Haerter, P J; Illes, P

    1994-01-01

    equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7514664

  2. Subsets of ATP-sensitive potassium channel (KATP) inhibitors increase gap junctional intercellular communication in metastatic cancer cell lines independent of SUR expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gap junctional intercellular communication (GJIC) is a process whereby cells share molecules and nutrients with each other by physical contact through cell membrane pores. In tumor cells, GJIC is often altered, suggesting that this process may be important in the context of cancer. Certain ion chan...

  3. The SLO3 sperm-specific potassium channel plays a vital role in male fertility

    PubMed Central

    Santi, Celia M; Martínez-López, Pablo; de la Vega-Beltrán, José Luis; Butler, Alice; Alisio, Arturo; Darszon, Alberto; Salkoff, Lawrence

    2010-01-01

    Here we show a unique example of male infertility conferred by a gene knock-out of the sperm-specific, pH-dependent SLO3 potassium channel. In striking contrast to wild-type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent “hairpin” shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation-induced hyperpolarization, and membrane hyperpolarization is crucial to the AR. PMID:20138882

  4. Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures.

    PubMed

    Harmon, C S; Lutz, D; Ducote, J

    1993-01-01

    We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

  5. Ceramide modulates HERG potassium channel gating by translocation into lipid rafts

    PubMed Central

    Ganapathi, Sindura B.; Fox, Todd E.; Elmslie, Keith S.

    2010-01-01

    Human ether-à-go-go-related gene (HERG) potassium channels play an important role in cardiac action potential repolarization, and HERG dysfunction can cause cardiac arrhythmias. However, recent evidence suggests a role for HERG in the proliferation and progression of multiple types of cancers, making it an attractive target for cancer therapy. Ceramide is an important second messenger of the sphingolipid family, which due to its proapoptotic properties has shown promising results in animal models as an anticancer agent. Yet the acute effects of ceramide on HERG potassium channels are not known. In the present study we examined the effects of cell-permeable C6-ceramide on HERG potassium channels stably expressed in HEK-293 cells. C6-ceramide (10 μM) reversibly inhibited HERG channel current (IHERG) by 36 ± 5%. Kinetically, ceramide induced a significant hyperpolarizing shift in the current-voltage relationship (ΔV1/2 = −8 ± 0.5 mV) and increased the deactivation rate (43 ± 3% for τfast and 51 ± 3% for τslow). Mechanistically, ceramide recruited HERG channels within caveolin-enriched lipid rafts. Cholesterol depletion and repletion experiments and mathematical modeling studies confirmed that inhibition and gating effects are mediated by separate mechanisms. The ceramide-induced hyperpolarizing gating shift (raft mediated) could offset the impact of inhibition (raft independent) during cardiac action potential repolarization, so together they may nullify any negative impact on cardiac rhythm. Our results provide new insights into the effects of C6-ceramide on HERG channels and suggest that C6-ceramide can be a promising therapeutic for cancers that overexpress HERG. PMID:20375276

  6. Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Ki...

  7. Activation of ATP-sensitive potassium channel by iptakalim normalizes stress-induced HPA axis disorder and depressive behaviour by alleviating inflammation and oxidative stress in mouse hypothalamus.

    PubMed

    Zhao, Xiao-Jie; Zhao, Zhan; Yang, Dan-Dan; Cao, Lu-Lu; Zhang, Ling; Ji, Juan; Gu, Jun; Huang, Ji-Ye; Sun, Xiu-Lan

    2017-02-01

    Stress-induced disturbance of the hypothalamic-pituitary-adrenal (HPA) axis is strongly implicated in incidence of mood disorders. A heightened neuroinflammatory response and oxidative stress play a fundamental role in the dysfunction of the HPA axis. We have previously demonstrated that iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could prevent oxidative injury and neuroinflammation against multiple stimuli-induced brain injury. The present study was to demonstrate the impacts of Ipt in stress-induced HPA axis disorder and depressive behavior. We employed 2 stress paradigms: 8 weeks of continuous restraint stress (chronic restraint stress, CRS) and 2h of restraint stress (acute restraint stress, ARS), to mimic both chronic stress and severe acute stress. Prolonged (4 weeks) and short-term (a single injection) Ipt treatment was administered 30min before each stress paradigm. We found that HPA axis was altered after stress, with different responses to CRS (lower ACTH and CORT, higher AVP, but normal CRH) and ARS (higher CRH, ACTH and CORT, but normal AVP). Both prolonged and short-term Ipt treatment normalized stress-induced HPA axis disorders and abnormal behaviors in mice. CRS and ARS up-regulated mRNA levels of inflammation-related molecules (TNFα, IL-1β, IL-6 and TLR4) and oxidative stress molecules (gp91phox, iNOS and Nrf2) in the mouse hypothalamus. Double immunofluorescence showed CRS and ARS increased microglia activation (CD11b and TNFα) and oxidative stress in neurons (NeuN and gp91phox), which were alleviated by Ipt. Therefore, the present study reveals that Ipt could prevent against stress-induced HPA axis disorders and depressive behavior by alleviating inflammation and oxidative stress in the hypothalamus.

  8. Altered Expression of Two-Pore Domain Potassium (K2P) Channels in Cancer

    PubMed Central

    Williams, Sarah; Bateman, Andrew; O'Kelly, Ita

    2013-01-01

    Potassium channels have become a focus in cancer biology as they play roles in cell behaviours associated with cancer progression, including proliferation, migration and apoptosis. Two-pore domain (K2P) potassium channels are background channels which enable the leak of potassium ions from cells. As these channels are open at rest they have a profound effect on cellular membrane potential and subsequently the electrical activity and behaviour of cells in which they are expressed. The K2P family of channels has 15 mammalian members and already 4 members of this family (K2P2.1, K2P3.1, K2P9.1, K2P5.1) have been implicated in cancer. Here we examine the expression of all 15 members of the K2P family of channels in a range of cancer types. This was achieved using the online cancer microarray database, Oncomine (www.oncomine.org). Each gene was examined across 20 cancer types, comparing mRNA expression in cancer to normal tissue. This analysis revealed all but 3 K2P family members (K2P4.1, K2P16.1, K2P18.1) show altered expression in cancer. Overexpression of K2P channels was observed in a range of cancers including breast, leukaemia and lung while more cancers (brain, colorectal, gastrointestinal, kidney, lung, melanoma, oesophageal) showed underexpression of one or more channels. K2P1.1, K2P3.1, K2P12.1, were overexpressed in a range of cancers. While K2P1.1, K2P3.1, K2P5.1, K2P6.1, K2P7.1 and K2P10.1 showed significant underexpression across the cancer types examined. This analysis supports the view that specific K2P channels may play a role in cancer biology. Their altered expression together with their ability to impact the function of other ion channels and their sensitivity to environmental stimuli (pO2, pH, glucose, stretch) makes understanding the role these channels play in cancer of key importance. PMID:24116006

  9. Altered expression of two-pore domain potassium (K2P) channels in cancer.

    PubMed

    Williams, Sarah; Bateman, Andrew; O'Kelly, Ita

    2013-01-01

    Potassium channels have become a focus in cancer biology as they play roles in cell behaviours associated with cancer progression, including proliferation, migration and apoptosis. Two-pore domain (K2P) potassium channels are background channels which enable the leak of potassium ions from cells. As these channels are open at rest they have a profound effect on cellular membrane potential and subsequently the electrical activity and behaviour of cells in which they are expressed. The K2P family of channels has 15 mammalian members and already 4 members of this family (K2P2.1, K2P3.1, K2P9.1, K2P5.1) have been implicated in cancer. Here we examine the expression of all 15 members of the K2P family of channels in a range of cancer types. This was achieved using the online cancer microarray database, Oncomine (www.oncomine.org). Each gene was examined across 20 cancer types, comparing mRNA expression in cancer to normal tissue. This analysis revealed all but 3 K2P family members (K2P4.1, K2P16.1, K2P18.1) show altered expression in cancer. Overexpression of K2P channels was observed in a range of cancers including breast, leukaemia and lung while more cancers (brain, colorectal, gastrointestinal, kidney, lung, melanoma, oesophageal) showed underexpression of one or more channels. K2P1.1, K2P3.1, K2P12.1, were overexpressed in a range of cancers. While K2P1.1, K2P3.1, K2P5.1, K2P6.1, K2P7.1 and K2P10.1 showed significant underexpression across the cancer types examined. This analysis supports the view that specific K2P channels may play a role in cancer biology. Their altered expression together with their ability to impact the function of other ion channels and their sensitivity to environmental stimuli (pO2, pH, glucose, stretch) makes understanding the role these channels play in cancer of key importance.

  10. Diet-induced obesity causes long QT and reduces transcription of voltage-gated potassium channels.

    PubMed

    Huang, Haiyan; Amin, Vaibhav; Gurin, Michael; Wan, Elaine; Thorp, Edward; Homma, Shunichi; Morrow, John P

    2013-06-01

    In humans, obesity is associated with long QT, increased frequency of premature ventricular complexes, and sudden cardiac death. The mechanisms of the pro-arrhythmic electrophysiologic remodeling of obesity are poorly understood. We tested the hypothesis that there is decreased expression of voltage-gated potassium channels in the obese heart, leading to long QT. Using implanted telemeters, we found that diet-induced obese (DIO) wild-type mice have impaired cardiac repolarization, demonstrated by long QT, as well as more frequent ventricular ectopy, similar to obese humans. DIO mice have reduced protein and mRNA levels of the potassium channel Kv1.5 caused by a reduction of the transcription factor cyclic AMP response element binding protein (CREB) in DIO hearts. We found that CREB knock-down by siRNA reduces Kv1.5, CREB binds to the Kv1.5 promoter in the heart, and CREB increases transcription of mouse and human Kv1.5 promoters. The reduction in CREB protein during lipotoxicity can be rescued by inhibiting protein kinase D (PKD). Our results identify a mechanism for obesity-induced electrophysiologic remodeling in the heart, namely PKD-induced reduction of CREB, which in turn decreases expression of the potassium channel Kv1.5.

  11. Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel.

    PubMed

    Akopova, O V; Nosar, V I; Mankovskaya, I N; Sagach, V F

    2008-10-01

    The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.

  12. Synthesis and Biological Evaluation of New Tricyclic Dihydropyridine Based Derivatives on Potassium Channels

    PubMed Central

    Gündüz, Miyase Gözde; Kaya, Yesim; Şimşek, Rahime; Sahin-Erdemli, Inci; Şafak, Cihat

    2016-01-01

    The present study reports a microwave-assisted method for the synthesis of twelve novel tricyclic 1,4-dihydropyridine derivatives in which dimethyl-substituted cyclohexane and / or tetrahydrothiophene rings are fused to the DHP ring. The structures of the compounds were confirmed by spectral methods and elemental analysis. The potassium channel opening effects of the compounds were determined on rat mesenteric arteries and urinary bladders. The obtained results indicated that some compounds produced mesenteric artery-selective relaxant properties and the effects of these compounds were mediated through ATP-sensitive potassium channels. The replacement of the second tetrahydrothiophene ring with dimethyl-substituted cyclohexane ring led to more active compounds. Docking studies were carried out to understand the interactions of the compounds with the active site of potassium channel. The unsubstituted nitrogen atom on the 1,4-dihydropyridine ring and one of the sulfonyl oxygens were found to be important for the formation of hydrogen bonds to stabilize the compound in the center of the cavity. The nature and position of phenyl ring substituents were also effective on the activity of the compounds. Finally, a theoretical study was established to predict the ADME of the most active compounds. PMID:28243272

  13. Molecular Information of charybdotoxin blockade in the large conductance calcium-activated potassium channel.

    PubMed

    Qiu, Su; Yi, Hong; Liu, Hui; Cao, Zhijian; Wu, Yingliang; Li, Wenxin

    2009-07-01

    The scorpion toxin, charybdotoxin (ChTX), is the first identified peptide inhibitor for the large-conductance Ca2+ and voltage-dependent K+ (BK) channel, and the chemical information of the interaction between ChTX and BK channel remains unclear today. Using combined computational methods, we obtained a ChTX-BK complex structure model, which correlated well with the mutagenesis data. In this complex, ChTX mainly used its beta-sheet domains to associate the BK channel with a conserved pore-blocking Lys27. Another crucial Tyr36 residue of ChTX lied over the loop connecting selectivity filter and S6 helix of BK channel, forming a hydrogen bond with Gly291 of BK channel. Besides, the unique turret region of BK channel was found to be far away from bound ChTX, which could explain the fact that many BK channel blockers show less selectivity over Kv channels. Together, all these information is helpful to reveal the diverse interactions between scorpion toxins and potassium channels and can accelerate the molecular engineering of specific inhibitor design.

  14. Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning

    PubMed Central

    Rathour, Rahul Kumar; Malik, Ruchi; Narayanan, Rishikesh

    2016-01-01

    Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor. PMID:27094086

  15. Fear conditioning suppresses large-conductance calcium-activated potassium channels in lateral amygdala neurons.

    PubMed

    Sun, P; Zhang, Q; Zhang, Y; Wang, F; Wang, L; Yamamoto, R; Sugai, T; Kato, N

    2015-01-01

    It was previously shown that depression-like behavior is accompanied with suppression of the large-conductance calcium activated potassium (BK) channel in cingulate cortex pyramidal cells. To test whether BK channels are also involved in fear conditioning, we studied neuronal properties of amygdala principal cells in fear conditioned mice. After behavior, we made brain slices containing the amygdala, the structure critically relevant to fear memory. The resting membrane potential in lateral amygdala (LA) neurons obtained from fear conditioned mice (FC group) was more depolarized than in neurons from naïve controls. The frequencies of spikes evoked by current injections were higher in neurons from FC mice, demonstrating that excitability of LA neurons was elevated by fear conditioning. The depolarization in neurons from FC mice was shown to depend on BK channels by using the BK channel blocker charybdotoxin. Suppression of BK channels in LA neurons from the FC group was further confirmed on the basis of the spike width, since BK channels affect the descending phase of spikes. Spikes were broader in the FC group than those in the naïve control in a manner dependent on BK channels. Consistently, quantitative real-time PCR revealed a decreased expression of BK channel mRNA. The present findings suggest that emotional disorder manifested in the forms of fear conditioning is accompanied with BK channel suppression in the amygdala, the brain structure critical to this emotional disorder.

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

    PubMed

    Tang, Xue; Shao, Juan; Qin, Xiaohong

    2016-08-01

    KCNH voltage-gated potassium channels play critical roles in regulating cellular functions. The channel is composed of four subunits, each of which contains six transmembrane helices forming the central pore. The cytoplasmic parts of the subunits present a Per-Arnt-Sim (PAS) domain at the N-terminus and a cyclic nucleotide-binding homology domain at the C-terminus. PAS domains are conserved from prokaryotes to eukaryotes and are involved in sensing signals and cellular responses. To better understand the functional roles of PAS domains in KCNH channels, the structure of this domain from the human ether-à-go-go channel (hEAG channel) was determined. By comparing it with the structures of the Homo sapiens EAG-related gene (hERG) channel and the Drosophila EAG-like K(+) (dELK) channel and analyzing the structural features of the hEAG channel, it was identified that a hydrophobic patch on the β-sheet may mediate interaction between the PAS domain and other regions of the channel to regulate its functions.

  17. The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

    PubMed Central

    Shahidullah, Mohammad; Covarrubias, Manuel

    2003-01-01

    Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb+ is an excellent tool for this purpose because its prolonged residency time in the pore delays K+ channel closing. The data showed that, only when Rb+ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K+ current). Furthermore, macroscopic Rb+ currents were larger than K+ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K+ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels. PMID:12547775

  18. Probing the cavity of the slow inactivated conformation of shaker potassium channels.

    PubMed

    Panyi, Gyorgy; Deutsch, Carol

    2007-05-01

    Slow inactivation involves a local rearrangement of the outer mouth of voltage-gated potassium channels, but nothing is known regarding rearrangements in the cavity between the activation gate and the selectivity filter. We now report that the cavity undergoes a conformational change in the slow-inactivated state. This change is manifest as altered accessibility of residues facing the aqueous cavity and as a marked decrease in the affinity of tetraethylammonium for its internal binding site. These findings have implications for global alterations of the channel during slow inactivation and putative coupling between activation and slow-inactivation gates.

  19. Ion conduction in the KcsA potassium channel analyzed with a minimal kinetic model.

    PubMed

    Mafé, Salvador; Pellicer, Julio

    2005-02-01

    We use a model by Nelson to study the current-voltage and conductance-concentration curves of bacterial potassium channel KcsA without assuming rapid ion translocation. Ion association to the channel filter is rate controlling at low concentrations, but dissociation and transport in the filter can limit conduction at high concentration for ions other than K+. The absolute values of the effective rate constants are tentative but the relative changes in these constants needed to qualitatively explain the experiments should be of significance.

  20. Human Slack potassium channel mutations increase positive cooperativity between individual channels

    PubMed Central

    Barcia, Giulia; Quraishi, Imran H.; Martin, Hilary C.; Blair, Edward; Taylor, Jenny C.; Dulac, Olivier; Colleaux, Laurence

    2015-01-01

    Summary Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation or voltage-dependence. We examined nine different mutations of the KCNT1 (Slack) Na+-activated K+ channel that give rise to three distinct forms of epilepsy. All produced many fold-increases in current amplitude over that of the wild type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gives rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of the mutations is to alter channel-channel interactions. PMID:25482562

  1. Human slack potassium channel mutations increase positive cooperativity between individual channels.

    PubMed

    Kim, Grace E; Kronengold, Jack; Barcia, Giulia; Quraishi, Imran H; Martin, Hilary C; Blair, Edward; Taylor, Jenny C; Dulac, Olivier; Colleaux, Laurence; Nabbout, Rima; Kaczmarek, Leonard K

    2014-12-11

    Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na(+)-activated K(+) channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions.

  2. Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

    PubMed Central

    Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

    2016-01-01

    KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001 PMID:27542194

  3. Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia.

    PubMed

    Selga, Elisabet; Pérez-Serra, Alexandra; Moreno-Asso, Alba; Anderson, Seth; Thomas, Kristen; Desai, Mayurika; Brugada, Ramon; Pérez, Guillermo J; Scornik, Fabiana S

    2013-01-01

    Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the β1 and β4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular β subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory β subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.

  4. Emerging concepts for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease

    PubMed Central

    Lüscher, Christian; Slesinger, Paul A.

    2010-01-01

    G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to the activation of many G-protein coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on their subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that GIRK activity has an important role in physiological responses, including pain perception and memory modulation. Moreover, abnormal GIRK function has been implicated in altering neuronal excitability and cell death that may be important in the pathophysiology of human diseases such as epilepsy, Down’s syndrome, Parkinson’s disease and drug addiction. GIRK channels may therefore prove to be a valuable new therapeutic target for treating these health problems. PMID:20389305

  5. hERG1 potassium channel in cancer cells: a tool to reprogram immortality.

    PubMed

    Gentile, Saverio

    2016-10-01

    It has been well established that changes in ion fluxes across cellular membranes as a function of time is fundamental in maintaining cellular homeostasis of every living cell. Consequently, dysregulation of ion channels activity is a critical event in pathological conditions of several tissues, including cancer. Nevertheless, the role of ion channels in cancer biology is still not well understood and very little is known about the possible therapeutic opportunities offered by the use of the vast collection of drugs that target ion channels. In this review, we focus on the recent advances in understanding the role of the voltage-gated hERG1 potassium channel in cancer and on the effects of pharmacologic manipulation of the hERG1 in cancer cells aiming to provide insights into the biochemical signaling and cellular processes that are altered by using these drugs.

  6. Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes

    PubMed Central

    Jarecki, Brian W.; Makino, Shin-ichi; Beebe, Emily T.; Fox, Brian G.; Chanda, Baron

    2013-01-01

    Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems. PMID:23301161

  7. Opening paths to novel analgesics: the role of potassium channels in chronic pain

    PubMed Central

    Tsantoulas, Christoforos; McMahon, Stephen B.

    2014-01-01

    Chronic pain is associated with abnormal excitability of the somatosensory system and remains poorly treated in the clinic. Potassium (K+) channels are crucial determinants of neuronal activity throughout the nervous system. Opening of these channels facilitates a hyperpolarizing K+ efflux across the plasma membrane that counteracts inward ion conductance and therefore limits neuronal excitability. Accumulating research has highlighted a prominent involvement of K+ channels in nociceptive processing, particularly in determining peripheral hyperexcitability. We review salient findings from expression, pharmacological, and genetic studies that have untangled a hitherto undervalued contribution of K+ channels in maladaptive pain signaling. These emerging data provide a framework to explain enigmatic pain syndromes and to design novel pharmacological treatments for these debilitating states. PMID:24461875

  8. Movement of the S4 segment in the hERG potassium channel during membrane depolarization.

    PubMed

    Elliott, David J S; Dondas, Naciye Y; Munsey, Tim S; Sivaprasadarao, Asipu

    2009-12-01

    The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly - a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.

  9. Potassium channels: how genetic studies of epileptic syndromes open paths to new therapeutic targets and drugs.

    PubMed

    Cooper, E C

    2001-01-01

    How can epilepsy gene hunting lead to better care for patients with epilepsy? Lessons may be learned from the progress made by identifying the mutated genes that cause Benign Familial Neonatal Convulsions (BFNC). In 1998, a decade of clinical and laboratory-based genetics work resulted in the cloning of the KCNQ2 potassium channel gene at the BFNC locus on chromosome 20. Subsequently, computer "mining" of public DNA databases allowed the rapid identification of three more brain KCNQ genes. Mutations in each of these additional genes were implicated as causes of human hereditary diseases: epilepsy (KCNQ3), deafness (KCNQ4), and, possibly, retinal degeneration (KCNQ5). Physiologists discovered that the KCNQ genes encoded subunits of the "M-channel," a type of potassium channel known to control repetitive neuronal discharges. Finally, pharmacologists discovered that retigabine, a novel anticonvulsant with a broad but distinctive efficacy profile in animal studies, was a potent KCNQ channel opener. These studies suggest that KCNQ channels may be an important new class of targets for anticonvulsant therapies. The efficacy of retigabine is currently being tested in multicenter clinical trials; identification of its molecular targets will allow it to be more efficiently exploited as a "lead compound." Cloned human KCNQ channels can now be expressed in cultured cells for "high-throughput" screening of drug candidates. Ongoing studies of the KCNQ channels in humans and animal models will refine our understanding of how M-channels control excitability at the cellular, network, and behavioral levels, and may reveal additional targets for therapeutic manipulation.

  10. Dendritic A-type potassium channel subunit expression in CA1 hippocampal interneurons.

    PubMed

    Menegola, M; Misonou, H; Vacher, H; Trimmer, J S

    2008-06-26

    Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and potassium channel interacting protein (KChIP) 1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal Ammon's horn (CA1) neurons. Immunohistochemistry was performed on 40 mum rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with a unique dendritic A-type channel complement and ability to control BAPs.

  11. Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

    PubMed Central

    Zhong, Lei Ray; Estes, Stephen; Artinian, Liana; Rehder, Vincent

    2013-01-01

    Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons. PMID:24236040

  12. Cloning and characterization of a human delayed rectifier potassium channel gene.

    PubMed

    Albrecht, B; Lorra, C; Stocker, M; Pongs, O

    1993-01-01

    A human genomic DNA library was screened for sequences homologues to the rat delayed rectifier Kv 2.1 (DRK1) K+ channel cDNA. Three phages were isolated which hybridized to Kv 2.1 cDNA probes. Alignment of the human genomic DNA sequence with the rat cDNA sequence indicated that the open reading frame (ORF) is interrupted by a large intervening sequence, that separates exons encoding the membrane spanning core region of the K+ channel polypeptide. The Kv 2.1 gene occurs once in the human genome and has been mapped to chromosome 20. The human, mouse and rat Kv 2.1 proteins have been highly conserved, showing only a few substitutions outside of the membrane spanning domains in the amino- and carboxy-terminal cytoplasmic domains. Nevertheless, expression of human DRK1 channels in Xenopus oocytes showed that mouse, rat and human Kv 2.1 channels have distinct pharmacological and electrophysiological properties. The observed differences in activation, voltage-dependence, 4-aminopyridine sensitivity and single-channel conductance have to be attributed to amino acid substitutions in the amino-and/or carboxy-terminal cytoplasmic domains. Obviously, these domains of Kv 2.1 channels influence biophysical K+ channel properties, which are thought to be determined solely by the membrane spanning core domain of potassium channels.

  13. Dendrotoxins: structure-activity relationships and effects on potassium ion channels.

    PubMed

    Harvey, A L; Robertson, B

    2004-12-01

    Dendrotoxins are small proteins isolated from mamba (Dendroaspis) snakes. The original dendrotoxin was found in venom of the Eastern green mamba, Dendroaspis angusticeps, and related proteins were subsequently found in other mamba venoms. The dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges, and they are homologous to Kunitz-type serine protease inhibitors, such as aprotinin (BPTI). The dendrotoxins have little or no anti-protease activity, but they block particular subtypes of voltage-dependent potassium channels of the Kv1 subfamily in neurones. Alpha-dendrotoxin from green mamba Dendroaspis angusticeps and toxin I from the black mamba Dendroaspis polylepis block cloned Kv1.1, Kv1.2 and Kv1.6 channels in the low nanomolar range; toxin K, also from the black mamba Dendroaspis polylepis, preferentially blocks Kv1.1 channels and is active at picomolar concentrations. Structural modifications and mutations to dendrotoxins have helped to define the molecular recognition properties of different types of K+ channels, although more work is needed to characterise the chemical features of the toxins that underlie their selectivity and potency at particular subtypes of channels. Dendrotoxins have been useful markers of subtypes of K+ channels in vivo, and dendrotoxins have become widely used as probes for studying the function of K+ channels in physiology and pathophysiology. With some pathological conditions being associated with voltage-gated K+ channels, analogues of dendrotoxins might have therapeutic potential.

  14. Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems

    PubMed Central

    Roy Chowdhury, Uttio; Bahler, Cindy K.; Holman, Bradley H.; Dosa, Peter I.; Fautsch, Michael P.

    2015-01-01

    Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development. PMID:26535899

  15. Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels

    PubMed Central

    Xia, Menghang; Shahane, Sampada; Huang, Ruili; Titus, Steven A.; Shum, Enoch; Zhao, Yong; Southall, Noel; Zheng, Wei; Witt, Kristine L.; Tice, Raymond R.; Austin, Christopher P.

    2011-01-01

    The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K+) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially lead to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC50 potencies ranging from 0.26 to 22 μM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC50 value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo. PMID:21362439

  16. Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels

    SciTech Connect

    Xia Menghang; Shahane, Sampada A.; Huang, Ruili; Titus, Steven A.; Shum, Enoch; Zhao Yong; Southall, Noel; Zheng, Wei; Witt, Kristine L.; Tice, Raymond R.; Austin, Christopher P.

    2011-05-01

    The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K{sup +}) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC{sub 50} potencies ranging from 0.26 to 22 {mu}M. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC{sub 50} value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.

  17. A novel crystallization method for visualizing the membrane localization of potassium channels.

    PubMed Central

    Lopatin, A N; Makhina, E N; Nichols, C G

    1998-01-01

    The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. PMID:9591643

  18. Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal

    PubMed Central

    Sun, Xiao-Ping; Schlichter, Lyanne C; Stanley, Elis F

    1999-01-01

    A high-conductance calcium-activated potassium channel (BK KCa) was characterized at a cholinergic presynaptic nerve terminal using the calyx synapse isolated from the chick ciliary ganglion.The channel had a conductance of 210 pS in a 150 mM:150 mM K+ gradient, was highly selective for K+ over Na+, and was sensitive to block by external charybdotoxin or tetraethylammonium (TEA) and by internal Ba2+. At +60 mV it was activated by cytoplasmic calcium [Ca2+]i with a Kd of ≈0.5 μM and a Hill coefficient of ≈2.0. At 10 μM [Ca2+]i the channel was 50 % activated (V½) at -8.0 mV with a voltage dependence (Boltzmann slope-factor) of 32.7 mV. The V½ values hyperpolarized with an increase in [Ca2+]i while the slope factors decreased. There were no overt differences in conductance or [Ca2+]i sensitivity between BK channels from the transmitter release face and the non-release face.Open and closed times were fitted by two and three exponentials, respectively. The slow time constants were strongly affected by both [Ca2+]i and membrane potential changes.In cell-attached patch recordings BK channel opening was enhanced by a prepulse permissive for calcium influx through the patch, suggesting that the channel can be activated by calcium ion influx through neighbouring calcium channels.The properties of the presynaptic BK channel are well suited for rapid activation during the presynaptic depolarization and Ca2+ influx that are associated with transmitter release. This channel may play an important role in terminating release by rapid repolarization of the action potential. PMID:10420003

  19. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

    PubMed

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-06-15

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

  20. Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel.

    PubMed

    Marques-Carvalho, Maria João; Oppermann, Johannes; Muñoz, Eva; Fernandes, Andreia S; Gabant, Guillaume; Cadene, Martine; Heinemann, Stefan H; Schönherr, Roland; Morais-Cabral, João Henrique

    2016-10-04

    The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca(2+)/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.

  1. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

    PubMed Central

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-01-01

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

  2. Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore

    PubMed Central

    Chokshi, Rikki H.; Larsen, Aaron T.; Bhayana, Brijesh

    2015-01-01

    Compounds PKTHPP (1-{1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl}propan-1-one), A1899 (2ʹ′-[(4-methoxybenzoylamino)methyl]biphenyl-2-carboxylic acid 2,4-difluorobenzylamide), and doxapram inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and stimulate breathing. To better understand the molecular mechanism(s) of action of these drugs, we undertook studies to identify amino acid residues in the TASK-3 protein that mediate this inhibition. Guided by homology modeling and molecular docking, we hypothesized that PKTHPP and A1899 bind in the TASK-3 intracellular pore. To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118–128 and 228–248), individually, to a negatively charged aspartate. We quantified each mutation's effect on TASK-3 potassium channel concentration response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. TASK-3 pore mutations at residues 122 (L122D, E, or K) and 236 (G236D) caused the IC50 of PKTHPP to increase more than 1000-fold. TASK-3 mutants L122D, G236D, L239D, and V242D were resistant to block by PKTHPP, A1899, and doxapram. Our data are consistent with a model in which breathing stimulant compounds PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding at a common site within the channel intracellular pore region, although binding outside the channel pore cannot yet be excluded. PMID:26268529

  3. Activation and inactivation of homomeric KvLQT1 potassium channels.

    PubMed Central

    Pusch, M; Magrassi, R; Wollnik, B; Conti, F

    1998-01-01

    The voltage-gated potassium channel protein KvLQT1 (Wang et al., 1996. Nature Genet. 12:17-23) is believed to underlie the delayed rectifier potassium current of cardiac muscle together with the small membrane protein minK (also named IsK) as an essential auxiliary subunit (Barhanin et al., 1996. Nature. 384:78-80; Sanguinetti et al., 1996. Nature. 384:80-83) Using the Xenopus oocyte expression system, we analyzed in detail the gating characteristics of homomeric KvLQT1 channels and of heteromeric KvLQT1/minK channels using two-electrode voltage-clamp recordings. Activation of homomeric KvLQT1 at positive voltages is accompanied by an inactivation process that is revealed by a transient increase in conductance after membrane repolarization to negative values. We studied the recovery from inactivation and the deactivation of the channels during tail repolarizations at -120 mV after conditioning pulses of variable amplitude and duration. Most measurements were made in high extracellular potassium to increase the size of inward tail currents. However, experiments in normal low-potassium solutions showed that, in contrast to classical C-type inactivation, the inactivation of KvLQT1 is independent of extracellular potassium. At +40 mV inactivation develops with a delay of 100 ms. At the same potential, the activation estimated from the amplitude of the late exponential decay of the tail currents follows a less sigmoidal time course, with a late time constant of 300 ms. Inactivation of KvLQT1 is not complete, even at the most positive voltages. The delayed, voltage-dependent onset and the incompleteness of inactivation suggest a sequential gating scheme containing at least two open states and ending with an inactivating step that is voltage independent. In coexpression experiments of KvLQT1 with minK, inactivation seems to be largely absent, although biphasic tails are also observed that could be related to similar phenomena. PMID:9675180

  4. Novel expression and regulation of voltage-dependent potassium channels in placentas from women with preeclampsia.

    PubMed

    Mistry, Hiten D; McCallum, Laura A; Kurlak, Lesia O; Greenwood, Iain A; Broughton Pipkin, Fiona; Tribe, Rachel M

    2011-09-01

    Preeclampsia is associated with structural/functional alterations in placental and maternal vasculature. Voltage-dependant potassium channels encoded by KCNQ1-5 genes have been detected in several types of blood vessels where they promote vascular relaxation. Voltage-dependant potassium channel function can be modulated by KCNE1-5-encoded accessory proteins. The aim of this study was to determine whether KCNQ and KCNE genes are differentially expressed in placentas from women with preeclampsia compared with normotensive controls and to examine any differences in those who delivered preterm (<37 weeks) or term. Placental biopsies (from midway between the cord and periphery) were obtained, with consent, from white European control (n=24; term) and preeclamptic (n=22; of whom 8 delivered before 37 weeks' gestation) women. KCNQ/KCNE and GAPDH mRNA expressions were determined by quantitative RT-PCR. Protein expression/localization was assessed using immunohistochemistry. KCNQ3 and KCNE5 mRNA expressions were significantly upregulated in preeclampsia (median [interquartile range]: 1.942 [0.905 to 3.379]) versus controls (0.159 [0.088 to 0.288]; P=0.001) and exhibited a strong positive correlation with each other (P<0.001), suggesting a novel heterodimer. Enhanced protein expression of KCNQ3 and KCNE5 in preeclampsia was confirmed with localization mainly restricted to the syncytiotrophoblast. KCNQ4 and KCNE1 isoforms were suppressed in placentas from term preeclamptic women versus controls (P≤0.05). KCNQ1 mRNA expression was increased and KCNQ5 decreased in the preterm preeclamptic group versus controls (P<0.05). In summary, voltage-dependant potassium channels are expressed and markedly modulated in placentas from preeclamptic women. Differential expression of isoforms may lead to altered cell proliferation. The correlation between KCNQ3 and KCNE5 expression is indicative of a novel channel complex and warrants further investigation.

  5. NMR structure of inactivation gates from mammalian voltage-dependent potassium channels.

    PubMed

    Antz, C; Geyer, M; Fakler, B; Schott, M K; Guy, H R; Frank, R; Ruppersberg, J P; Kalbitzer, H R

    1997-01-16

    The electrical signalling properties of neurons originate largely from the gating properties of their ion channels. N-type inactivation of voltage-gated potassium (Kv) channels is the best-understood gating transition in ion channels, and occurs by a 'ball-and-chain' type mechanism. In this mechanism an N-terminal domain (inactivation gate), which is tethered to the cytoplasmic side of the channel protein by a protease-cleavable chain, binds to its receptor at the inner vestibule of the channel, thereby physically blocking the pore. Even when synthesized as a peptide, ball domains restore inactivation in Kv channels whose inactivation domains have been deleted. Using high-resolution nuclear magnetic resonance (NMR) spectroscopy, we analysed the three-dimensional structure of the ball peptides from two rapidly inactivating mammalian K. channels (Raw3 (Kv3.4) and RCK4 (Kv1.4)). The inactivation peptide of Raw3 (Raw3-IP) has a compact structure that exposes two phosphorylation sites and allows the formation of an intramolecular disulphide bridge between two spatially close cysteine residues. Raw3-IP exhibits a characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region. The RCK4 inactivation peptide (RCK4-IP) shows a similar spatial distribution of charged and uncharged regions, but is more flexible and less ordered in its amino-terminal part.

  6. Gating motions in voltage-gated potassium channels revealed by coarse-grained molecular dynamics simulations.

    PubMed

    Treptow, Werner; Marrink, Siewert-J; Tarek, Mounir

    2008-03-20

    Voltage-gated potassium (Kv) channels are ubiquitous transmembrane proteins involved in electric signaling of excitable tissues. A fundamental property of these channels is the ability to open or close in response to changes in the membrane potential. To date, their structure-based activation mechanism remains unclear, and there is a large controversy on how these gates function at the molecular level, in particular, how movements of the voltage sensor domain are coupled to channel gating. So far, all mechanisms proposed for this coupling are based on the crystal structure of the open voltage-gated Kv1.2 channel and structural models of the closed form based on electrophysiology experiments. Here, we use coarse-grain (CG) molecular dynamics simulations that allow conformational changes from the open to the closed form of the channel (embedded in its membrane environment) to be followed. Despite the low specificity of the CG force field, the obtained closed structure satisfies several experimental constraints. The overall results suggest a gating mechanism in which a lateral displacement the S4-S5 linker leads to a closing of the gate. Only a small up-down movement of the S4 helices is noticed. Additionally, the study suggests a peculiar upward motion of the intracellular tetramerization domain of the channel, hence providing a molecular view on how this domain may further regulate conduction in Kv channels.

  7. Potassium Channels in Peripheral Pain Pathways: Expression, Function and Therapeutic Potential

    PubMed Central

    Du, Xiaona; Gamper, Nikita

    2013-01-01

    Electrical excitation of peripheral somatosensory nerves is a first step in generation of most pain signals in mammalian nervous system. Such excitation is controlled by an intricate set of ion channels that are coordinated to produce a degree of excitation that is proportional to the strength of the external stimulation. However, in many disease states this coordination is disrupted resulting in deregulated peripheral excitability which, in turn, may underpin pathological pain states (i.e. migraine, neuralgia, neuropathic and inflammatory pains). One of the major groups of ion channels that are essential for controlling neuronal excitability is potassium channel family and, hereby, the focus of this review is on the K+ channels in peripheral pain pathways. The aim of the review is threefold. First, we will discuss current evidence for the expression and functional role of various K+ channels in peripheral nociceptive fibres. Second, we will consider a hypothesis suggesting that reduced functional activity of K+ channels within peripheral nociceptive pathways is a general feature of many types of pain. Third, we will evaluate the perspectives of pharmacological enhancement of K+ channels in nociceptive pathways as a strategy for new analgesic drug design. PMID:24396338

  8. Biomimetic membrane platform containing hERG potassium channel and its application to drug screening.

    PubMed

    Arslan Yildiz, Ahu; Kang, CongBao; Sinner, Eva-Kathrin

    2013-04-07

    The hERG (human ether-à-go-go-related gene) potassium channel has been extensively studied by both academia and industry because of its relation to inherited or drug-induced long QT syndrome (LQTS). Unpredicted hERG and drug interaction affecting channel activity is of main concern for drug discovery. Although there are several methods to test hERG and drug interaction, it is still necessary to develop some efficient and economic ways to probe hERG and drug interactions. To contribute this aim, we have developed a biomimetic lipid membrane platform into which the hERG channel can be folded. Expression and integration of the hERG channel was achieved using a cell-free (CF) expression system. The folding of hERG in the biomimetic membrane system was investigated using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) and Imaging Surface Plasmon Resonance (iSPR). In addition, the hERG channel folded into our biomimetic membrane platform was used for probing the channel and drug interactions through fluorescence polarization (FP) assay. Our results suggest that the biomimetic system employed is capable of detecting the interaction between hERG and different channel blockers at varied concentrations. We believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions.

  9. Carboxy-terminal domain mediates assembly of the voltage-gated rat ether-à-go-go potassium channel.

    PubMed Central

    Ludwig, J; Owen, D; Pongs, O

    1997-01-01

    The specific assembly of subunits to oligomers is an important prerequisite for producing functional potassium channels. We have studied the assembly of voltage-gated rat ether-à-go-go (r-eag) potassium channels with two complementary assays. In protein overlay binding experiments it was shown that a 41-amino-acid domain, close to the r-eag subunit carboxy-terminus, is important for r-eag subunit interaction. In an in vitro expression system it was demonstrated that r-eag subunits lacking this assembly domain cannot form functional potassium channels. Also, a approximately 10-fold molar excess of the r-eag carboxy-terminus inhibited in co-expression experiments the formation of functional r-eag channels. When the r-eag carboxy-terminal assembly domain had been mutated, the dominant-negative effect of the r-eag carboxy-terminus on r-eag channel expression was abolished. The results demonstrate that a carboxy-terminal assembly domain is essential for functional r-eag potassium channel expression, in contrast to the one of Shaker-related potassium channels, which is directed by an amino-terminal assembly domain. PMID:9400421

  10. Contributions of counter-charge in a potassium channel voltage-sensor domain.

    PubMed

    Pless, Stephan A; Galpin, Jason D; Niciforovic, Ana P; Ahern, Christopher A

    2011-07-24

    Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly coevolved acidic and aromatic side chains assist the transfer of cationic side chains across the transmembrane electric field during voltage sensing. We investigated the functional contribution of negative electrostatic potentials from these residues to channel gating and voltage sensing with unnatural amino acid mutagenesis, electrophysiology, voltage-clamp fluorometry and ab initio calculations. The data show that neutralization of two conserved acidic side chains in transmembrane segments S2 and S3, namely Glu293 and Asp316 in Shaker potassium channels, has little functional effect on conductance-voltage relationships, although Glu293 appears to catalyze S4 movement. Our results suggest that neither Glu293 nor Asp316 engages in electrostatic state-dependent charge-charge interactions with S4, likely because they occupy, and possibly help create, a water-filled vestibule.

  11. PKC and AMPK regulation of Kv1.5 potassium channels

    PubMed Central

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi; Petersen, Frederic; MacAulay, Nanna; Rasmussen, Hanne Borger; Jespersen, Thomas

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K+ current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems. PMID:26043299

  12. Ethanol Affects Network Activity in Cultured Rat Hippocampus: Mediation by Potassium Channels

    PubMed Central

    Korkotian, Eduard; Bombela, Tatyana; Odegova, Tatiana; Zubov, Petr; Segal, Menahem

    2013-01-01

    The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25–0.5%) ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor. PMID:24260098

  13. Potassium Channel Block and Novel Autoimmune-Associated Long QT Syndrome.

    PubMed

    Boutjdir, Mohamed; Lazzerini, Pietro Enea; Capecchi, Pier Leopoldo; Laghi-Pasini, Franco; El-Sherif, Nabil

    2016-06-01

    This article reviews advances in the pathogenesis of anti-SSA/Ro antibody-induced corrected QT (QTc) prolongation in patients with autoimmune diseases; particularly connective tissue disease (CTD). Evidence shows that anti-SSA/Ro antibody-positive patients with CTD show QTc prolongation and complex ventricular arrhythmias. Molecular and functional data provide evidence that the human ether-a-go-go-related gene potassium channel conducting the rapidly activating delayed rectifier potassium current is directly inhibited by anti-SSA/Ro antibodies, resulting in action potential duration prolongation leading to QT interval lengthening. Routine electrocardiogram screening in anti-SSA/Ro antibody-positive patients and counseling for patients with other QTc prolonging risk factors is recommended.

  14. Barium, TEA and sodium sensitive potassium channels are present in the human placental syncytiotrophoblast apical membrane.

    PubMed

    Díaz, P; Vallejos, C; Guerrero, I; Riquelme, G

    2008-10-01

    The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno-fetal exchange. There is ample evidence suggesting the presence of potassium (K(+)) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K(+) currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K(+) channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba(+2)). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba(+2) respectively. The more frequent conductance was approximately 73pS, however several levels of current were detected suggesting the presence of more than one type of K(+) channel. In addition, sodium (Na(+)) sensitivity was detected in the patch current thus, over 10 mmol/L Na(+) reduced the seal current to 38%. These results were corroborated by the total current experiments where the K(+) current elicited in injected oocytes with apical purified membrane was blocked by Ba(+2) and TEA. The total current was also affected by Na(+), becoming larger when a Na(+)-free solution was used. Our results show the existence of at least two types of Ba(+2)-sensitive K(+) channels including a TEA sensitive sub-population, and some of them Na(+) sensitive K(+) channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K(+) channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.

  15. Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

    PubMed

    Linta, Leonhard; Boeckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2013-07-01

    The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

  16. Regional expression of the anesthetic-activated potassium channel TRESK in the rat nervous system

    PubMed Central

    Yoo, SieHyeon; Liu, Jia; Sabbadini, Marta; Au, Paul; Xie, Guo-xi; Yost, C. Spencer

    2009-01-01

    The two-pore-domain potassium (K2P) channels contribute to background (leak) potassium currents maintaining the resting membrane potential to play an important role in regulating neuronal excitability. As such they may contribute to nociception and the mechanism of action of volatile anesthetics. In the present study, we examined the protein expression pattern of the K2P channel TRESK in the rat central nervous system (CNS) and peripheral nervous system (PNS) by immunohistochemistry. The regional distribution expression pattern of TRESK has both similarities and significant differences from that of other K2P channels expressed in the CNS. TRESK expression is broadly found in the brain, spinal cord and dorsal root ganglia (DRG). TRESK expression is highest in important CNS structures, such as specific cortical layers, periaqueductal gray (PAG), granule cell layer of the cerebellum, and dorsal horn of the spinal cord. TRESK expression is also high in small and medium sized DRG neurons. These results provide an anatomic basis for identifying functional roles of TRESK in the rat nervous system. PMID:19716403

  17. The MiRP2-Kv3.4 potassium channel: muscling in on Alzheimer's disease.

    PubMed

    Choi, Eun; Abbott, Geoffrey W

    2007-09-01

    In this issue of Molecular Pharmacology (p. 665), Pannacione et al. provide evidence of a role for the voltage-gated potassium channel alpha subunit Kv3.4 and its ancillary subunit MiRP2 in beta-amyloid (Abeta) peptide-mediated neuronal death. The MiRP2-Kv3.4 channel complex-previously found to be important in skeletal myocyte physiology-is now argued to be a molecular correlate of the transient outward potassium current up-regulated by Abeta peptide, considered a significant step in the etiology of Alzheimer's disease. The authors conclude that MiRP2 and Kv3.4 are up-regulated by Abeta peptide in a nuclear factor kappaB-dependent fashion at the transcriptional level, and the sea anemone toxin BDS-I is shown to protect against Abeta peptide-mediated cell death by specific blockade of Kv3.4-generated current. The findings lend weight to the premise that specific channels, such as MiRP2-Kv3.4, could hold promise as future therapeutic targets in Alzheimer's disease and potentially other neurodegenerative disorders.

  18. Late cardiac preconditioning by exercise in dogs is mediated by mitochondrial potassium channels.

    PubMed

    Parra, Víctor M; Macho, Pilar; Domenech, Raúl J

    2010-09-01

    We previously showed that exercise induces myocardial preconditioning in dogs and that early preconditioning is mediated through mitochondrial adenosine triphosphate-sensitive potassium channels. We decided to study if late preconditioning by exercise is also mediated through these channels. Forty-eight dogs, surgically instrumented and trained to run daily, were randomly assigned to 4 groups: (1) Nonpreconditioned dogs: under anesthesia, the coronary artery was occluded during 1 hour and then reperfused during 4.5 hours. (2) Late preconditioned dogs: similar to group 1, but the dogs run on the treadmill for 5 periods of 5 minutes each, 24 hours before the coronary occlusion. (3) Late preconditioned dogs plus 5-hydroxydecanoate (5HD): similar to group 2, but 5HD was administered before the coronary occlusion. (4) Nonpreconditioned dogs plus 5HD: similar to group 1, but 5HD was administered before the coronary occlusion. Infarct size (percent of the risk region) decreased by effect of exercise by 56% (P < 0.05), and this effect was abolished with 5HD. 5HD by itself did not modify infarct size. Exercise did not induce myocardial ischemia, and the hemodynamics during ischemia-reperfusion period did not differ among groups. These effects were independent of changes in collateral flow to the ischemic region. We concluded that late cardiac preconditioning by exercise is mediated through mitochondrial adenosine triphosphate-sensitive potassium channels.

  19. Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization

    PubMed Central

    Auffenberg, Eva; Jurik, Angela; Mattusch, Corinna; Stoffel, Rainer; Genewsky, Andreas; Namendorf, Christian; Schmid, Roland M.; Rammes, Gerhard; Biel, Martin; Uhr, Manfred; Moosmang, Sven; Michalakis, Stylianos; Wotjak, Carsten T.; Thoeringer, Christoph K.

    2016-01-01

    Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research. PMID:26757616

  20. Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation

    NASA Astrophysics Data System (ADS)

    Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I.; Lindahl, Erik; Elinder, Fredrik

    2016-06-01

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions – a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

  1. Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels.

    PubMed Central

    Pusch, M; Bertorello, L; Conti, F

    2000-01-01

    The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in

  2. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

    NASA Astrophysics Data System (ADS)

    Lange, Adam; Giller, Karin; Hornig, Sönke; Martin-Eauclaire, Marie-France; Pongs, Olaf; Becker, Stefan; Baldus, Marc

    2006-04-01

    The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane-similar to the catalytic function of the active site of an enzyme-and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

  3. The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel

    PubMed Central

    Yazdi, Samira; Stein, Matthias; Elinder, Fredrik; Andersson, Magnus; Lindahl, Erik

    2016-01-01

    Voltage-gated potassium (KV) channels are membrane proteins that respond to changes in membrane potential by enabling K+ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs) induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD), the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker KV channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA) and the Shaker KV channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins. PMID:26751683

  4. Scorpion Toxins Specific for Potassium (K+) Channels: A Historical Overview of Peptide Bioengineering

    PubMed Central

    Bergeron, Zachary L.; Bingham, Jon-Paul

    2012-01-01

    Scorpion toxins have been central to the investigation and understanding of the physiological role of potassium (K+) channels and their expansive function in membrane biophysics. As highly specific probes, toxins have revealed a great deal about channel structure and the correlation between mutations, altered regulation and a number of human pathologies. Radio- and fluorescently-labeled toxin isoforms have contributed to localization studies of channel subtypes in expressing cells, and have been further used in competitive displacement assays for the identification of additional novel ligands for use in research and medicine. Chimeric toxins have been designed from multiple peptide scaffolds to probe channel isoform specificity, while advanced epitope chimerization has aided in the development of novel molecular therapeutics. Peptide backbone cyclization has been utilized to enhance therapeutic efficiency by augmenting serum stability and toxin half-life in vivo as a number of K+-channel isoforms have been identified with essential roles in disease states ranging from HIV, T-cell mediated autoimmune disease and hypertension to various cardiac arrhythmias and Malaria. Bioengineered scorpion toxins have been monumental to the evolution of channel science, and are now serving as templates for the development of invaluable experimental molecular therapeutics. PMID:23202307

  5. Basolateral potassium (IKCa) channel inhibition prevents increased colonic permeability induced by chemical hypoxia.

    PubMed

    Loganathan, A; Linley, J E; Rajput, I; Hunter, M; Lodge, J P A; Sandle, G I

    2011-01-01

    Major liver resection is associated with impaired intestinal perfusion and intestinal ischemia, resulting in decreased mucosal integrity, increased bacterial translocation, and an increased risk of postoperative sepsis. However, the mechanism by which ischemia impairs intestinal mucosal integrity is unclear. We therefore evaluated the role of Ca(2+)-sensitive, intermediate-conductance (IK(Ca)) basolateral potassium channels in enhanced intestinal permeability secondary to chemical hypoxia. The effects of chemical hypoxia induced by 100 μM dinitrophenol (DNP) and 5 mM deoxyglucose (DG) on basolateral IK(Ca) channel activity and whole cell conductance in intact human colonic crypts, and paracellular permeability (G(S)) in isolated colonic sheets, were determined by patch-clamp recording and transepithelial electrical measurements, respectively. DNP and DG rapidly stimulated IK(Ca) channels in cell-attached basolateral membrane patches and elicited a twofold increase (P = 0.004) in whole cell conductance in amphotericin B-permeabilized membrane patches, changes that were inhibited by the specific IK(Ca) channel blockers TRAM-34 (100 nM) and clotrimazole (CLT; 10 μM). In colonic sheets apically permeabilized with nystatin, DNP elicited a twofold increase (P = 0.005) in G(S), which was largely inhibited by the serosal addition of 50 μM CLT. We conclude that, in intestinal epithelia, chemical hypoxia increases G(S) through a mechanism involving basolateral IK(Ca) channel activation. Basolateral IK(Ca) channel inhibition may prevent or limit increased intestinal permeability during liver surgery.

  6. Molecular mechanism of pH sensing in KcsA potassium channels

    PubMed Central

    Thompson, Ameer N.; Posson, David J.; Parsa, Pirooz V.; Nimigean, Crina M.

    2008-01-01

    The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the proton-binding sites and at neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening. PMID:18443286

  7. The sodium-activated potassium channel Slack is required for optimal cognitive flexibility in mice.

    PubMed

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

    2015-07-01

    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 development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels.

  8. The sodium-activated potassium channel Slack is required for optimal cognitive flexibility in mice

    PubMed Central

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

    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 development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels. PMID:26077685

  9. Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels*

    PubMed Central

    Thomson, Steven J.; Hansen, Angela; Sanguinetti, Michael C.

    2015-01-01

    Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na+]i (e.g. during ischemia). An intracellular Na+ coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na+ coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na+ sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 m NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]i of 70 mm. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na+. In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mm niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]i from 3 to 100 mm. Thus, relative to fenamates, intracellular Na+ is a poor activator of Slo2.1. PMID:25903137

  10. Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels.

    PubMed

    Thomson, Steven J; Hansen, Angela; Sanguinetti, Michael C

    2015-06-05

    Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na(+)]i (e.g. during ischemia). An intracellular Na(+) coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na(+) coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na(+) sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 M NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]i of 70 mM. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na(+). In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mM niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]i from 3 to 100 mM. Thus, relative to fenamates, intracellular Na(+) is a poor activator of Slo2.1.

  11. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

    PubMed Central

    Schumacher, Jennifer A.; Wang, Xiaohong; Merrill, Sean A.; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M.; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons. PMID:26771544

  12. Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels.

    PubMed

    Murbartián, Janet; Lei, Qiubo; Sando, Julianne J; Bayliss, Douglas A

    2005-08-26

    Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

  13. Large-conductance Ca²⁺-activated potassium channel in mitochondria of endothelial EA.hy926 cells.

    PubMed

    Bednarczyk, Piotr; Koziel, Agnieszka; Jarmuszkiewicz, Wieslawa; Szewczyk, Adam

    2013-06-01

    In the present study, we describe the existence of a large-conductance Ca²⁺-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca²⁺, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β₂-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

  14. Nonlinearity of a Voltage-Gated Potassium Channel Revealed by the Mechanical Susceptibility

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Zocchi, Giovanni

    2013-01-01

    The voltage-gated potassium channel from Aeropyrum pernix operates by coupling the voltage-driven motion of a charged group of amino acids to the opening and closing of the pore. In this experiment, we drive this charged group with an ac field and observe the effect on the gating. The measurements for different frequencies and amplitudes of the forcing reveal an essential nonlinearity in the mechanical behavior of the molecule. Within a continuum-mechanics description, we extract the effective dissipation parameter γ for this conformational motion and find γ≈0.2g/s, similar to recent nanorheology measurements on the conformational motion of an enzyme.

  15. Eag1 Voltage-Dependent Potassium Channels: Structure, Electrophysiological Characteristics, and Function in Cancer.

    PubMed

    Wang, Xuzhao; Chen, Yafei; Zhang, Yuhong; Guo, Shuai; Mo, Li; An, Hailong; Zhan, Yong

    2017-02-03

    Eag1 (ether-à-go-go-1), a member of the voltage-dependent potassium channel family, is expressed mainly in the brain, and at low levels in placenta, testis, and adrenal gland, and only transiently in myoblasts. Recently, several studies have suggested that Eag1 is selectively expressed in various tumor tissues. Eag1 plays important roles in tumor proliferation, malignant transformation, invasion, metastasis, recurrence, and prognosis. Therefore, it has become a new molecular target for tumor diagnosis, prognosis evaluation, and tumor-targeted therapy. This review provides information about the current progress in understanding Eag1 structure, electrophysiological characteristics, and role in cancer.

  16. Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes*

    PubMed Central

    Jara-Oseguera, Andrés; Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Espinosa-Jalapa, Noel; Pérez-Guzmán, José A.; Elías-Viñas, David; Le Lagadec, Ronan; Rosenbaum, Tamara; Islas, León D.

    2011-01-01

    The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling. PMID:21454671

  17. Uncoupling charge movement from channel opening in voltage-gated potassium channels by ruthenium complexes.

    PubMed

    Jara-Oseguera, Andrés; Ishida, Itzel G; Rangel-Yescas, Gisela E; Espinosa-Jalapa, Noel; Pérez-Guzmán, José A; Elías-Viñas, David; Le Lagadec, Ronan; Rosenbaum, Tamara; Islas, León D

    2011-05-06

    The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K(+)-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K(+)-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

  18. The effect of ATP-sensitive potassium channel modulation on heart rate in isolated muskrat and guinea pig hearts.

    PubMed

    Streeby, D R; McKean, T A

    1994-12-01

    Muskrats (Ondontra zibethicus) are common freshwater diving mammals exhibiting a bradycardia with both forced and voluntary diving. This bradycardia is mediated by vagal innervation; however, if hypoxia is present there may be local factors that also decrease heart rate. Some of these local factors may include ATP-sensitive potassium channel activation and extracellular accumulation of potassium ions, hydrogen ions and lactate. The purpose of this study was to investigate the role of these factors in the isolated perfused hearts of muskrats and of a non-diving mammal, the guinea pig. Although lactate and proton administration reduced heart rate in isolated muskrat and guinea pig hearts, there was no difference in the response to lactate and proton infusion between the two species. Muskrat hearts were more sensitive to the heart-rate-lowering effects of exogenously applied potassium than were guinea pig hearts. Early increases in extracellular potassium concentration during hypoxia are thought to be mediated by the ATP-sensitive potassium channel. Activation of these channels under normoxic conditions had a mildly negative chronotropic effect in both species; however, activation of these channels with Lemakalim under hypoxic conditions caused the guinea pig heart to respond with an augmented bradycardia similar to that seen in the hypoxic muskrat heart in the absence of drugs. Inhibition of these channels by glibenclamide during hypoxia was partially successful in blocking the bradycardia in guinea pig hearts, but inhibition of the same channels in hypoxic muskrat hearts had a damaging effect as two of five hearts went into contracture during the hypoxia. Thus, although ATP-sensitive potassium channels appear to have a major role in the bradycardia of hypoxia in guinea pigs, the failure to prevent the bradycardia by inhibition of these channels in muskrat hearts suggests that multiple factors are involved in the hypoxia-induced bradycardia in this species.

  19. Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating

    PubMed Central

    Martin, Gregory M; Yoshioka, Craig; Rex, Emily A; Fay, Jonathan F; Xie, Qing; Whorton, Matthew R; Chen, James Z; Shyng, Show-Ling

    2017-01-01

    KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6 Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity. DOI: http://dx.doi.org/10.7554/eLife.24149.001 PMID:28092267

  20. Acepromazine inhibits hERG potassium ion channels expressed in human embryonic kidney 293 cells

    PubMed Central

    Joo, Young Shin; Lee, Hong Joon; Choi, Jin-Sung

    2017-01-01

    The effects of acepromazine on human ether-à-go-go-related gene (hERG) potassium channels were investigated using whole-cell voltage-clamp technique in human embryonic kidney (HEK293) cells transfected with hERG. The hERG currents were recorded with or without acepromazine, and the steady-state and peak tail currents were analyzed for the evaluating the drug effects. Acepromazine inhibited the hERG currents in a concentration-dependent manner with an IC50 value of 1.5 µM and Hill coefficient of 1.1. Acepromazine blocked hERG currents in a voltage-dependent manner between –40 and +10 mV. Before and after application of acepromazine, the half activation potentials of hERG currents changed to hyperpolarizing direction. Acepromazine blocked both the steady-state hERG currents by depolarizing pulse and the peak tail currents by repolarizing pulse; however, the extent of blocking by acepromazine in the repolarizing pulse was more profound than that in the depolarizing pulse, indicating that acepromazine has a high affinity for the open state of the channels, with a relatively lower affinity for the closed state of hERG channels. A fast application of acepromazine during the tail currents inhibited the open state of hERG channels in a concentration-dependent. The steady-state inactivation of hERG currents shifted to the hyperpolarized direction by acepromazine. These results suggest that acepromazine inhibits the hERG channels probably by an open- and inactivated-channel blocking mechanism. Regarding to the fact that the hERG channels are the potential target of drug-induced long QT syndrome, our results suggest that acepromazine can possibly induce a cardiac arrhythmia through the inhibition of hERG channels. PMID:28066143

  1. Identification of yeast proteins necessary for cell-surface function of a potassium channel.

    PubMed

    Haass, Friederike A; Jonikas, Martin; Walter, Peter; Weissman, Jonathan S; Jan, Yuh-Nung; Jan, Lily Y; Schuldiner, Maya

    2007-11-13

    Inwardly rectifying potassium (Kir) channels form gates in the cell membrane that regulate the flow of K(+) ions into and out of the cell, thereby influencing the membrane potential and electrical signaling of many cell types, including neurons and cardiomyocytes. Kir-channel function depends on other cellular proteins that aid in the folding of channel subunits, assembly into tetrameric complexes, trafficking of quality-controlled channels to the plasma membrane, and regulation of channel activity at the cell surface. We used the yeast Saccharomyces cerevisiae as a model system to identify proteins necessary for the functional expression of a mammalian Kir channel at the cell surface. A screen of 376 yeast strains, each lacking one nonessential protein localized to the early secretory pathway, identified seven deletion strains in which functional expression of the Kir channel at the plasma membrane was impaired. Six deletions were of genes with known functions in trafficking and lipid biosynthesis (sur4Delta, csg2Delta, erv14Delta, emp24Delta, erv25Delta, and bst1Delta), and one deletion was of an uncharacterized gene (yil039wDelta). We provide genetic and functional evidence that Yil039wp, a conserved, phosphoesterase domain-containing protein, which we named "trafficking of Emp24p/Erv25p-dependent cargo disrupted 1" (Ted1p), acts together with Emp24p/Erv25p in cargo exit from the endoplasmic reticulum (ER). The seven yeast proteins identified in our screen likely impact Kir-channel functional expression at the level of vesicle budding from the ER and/or the local lipid environment at the plasma membrane.

  2. The effect of intracellular anions on ATP-dependent potassium channels of rat skeletal muscle.

    PubMed Central

    McKillen, H C; Davies, N W; Stanfield, P R; Standen, N B

    1994-01-01

    1. We have used excised inside-out patches to study the effects of anions bathing the cytoplasmic surface of the membrane on ATP-dependent K+ channels of rat flexor digitorum brevis muscle. Channels were closed by ATP applied to the cytoplasmic face of the patch with a concentration for half-closure (Ki) of 14 microM, were highly selective for K+ and had unitary conductances of 62 pS in symmetrical 155 mM K+ and 27 pS in 5 mM [K+]o. 2. In 139 mM Cl- internal solution channel activity declined rapidly after excision of the patch. Inclusion of 40 mM potassium gluconate (substituted for KCl) in the solution both restored channel activity and greatly slowed its subsequent run-down. 3. The action of gluconate was concentration dependent. The effect did not involve a change in ATP binding, since the Ki for ATP was not significantly changed by gluconate, and was specific for the cytoplasmic face of the patch. 4. The anions pyruvate, lactate and acetate were all able to restore channel activity after run-down, though less well than gluconate, while sulphate and methylsulphate were without effect. 5. Analysis of single channel kinetics showed that gluconate did not affect mean open lifetime, but led to a decrease in the number and duration of long closings. 6. Anions are most likely to act by stabilizing the structure of the channel protein. Changes in the intracellular concentration of certain anions may play a role in regulating channel activity. PMID:7837093

  3. Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1)

    PubMed Central

    Tytgat, J; Maertens, Ch; Daenens, P

    1997-01-01

    Fluoxetine (Prozac) is widely used as an antidepressant drug and is assumed to be a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI). Claims that its beneficial psychotropic effects extend beyond those in treatment of depression have drawn clinical and popular attention to this compound, raising the question of whether there is anything exceptional about the supposed selective actions.We have used the voltage clamp technique to study the effect of fluoxetine on a neuronal, voltage-dependent potassium (K+) channel (RCK1; Kv1.1), expressed in Xenopus laevis oocytes. This channel subunit is abundantly expressed in the central nervous system and K+ channels containing this subunit are involved in the repolarization process of many types of neurones.Blockade of the K+ currents by fluoxetine was found to be use- and dose-dependent. Wash-out of this compound could not be achieved. Fluoxetine did not affect the ion selectivity of this K+ channel, as the reversal potential was unaltered.Slowing of both activation and deactivation kinetics of the channel by fluoxetine was observed, including tail current crossover upon repolarization.Hodgkin-Huxley type of models and more generalized Markov chain models were used to fit the kinetics of the data. Based upon a Markov kinetic scheme, our data can be interpreted to mean that blockade of fluoxetine consists of two components: a voltage-independent occurring in the last closed, but available state of the channel, and a voltage-dependent occurring in the open state.This study describes the first biophysical working model for the mechanism of action of fluoxetine on a neuronal, voltage-dependent K+ channel, RCK1. Although this channel is not very potently blocked by fluoxetine when expressed in oocytes, this study may help us to understand some of the clinical symptoms seen with elevated serum concentrations of this SSRI. PMID:9421290

  4. BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus.

    PubMed

    Raffaelli, Giacomo; Saviane, Chiara; Mohajerani, Majid H; Pedarzani, Paola; Cherubini, Enrico

    2004-05-15

    Large conductance calcium- and voltage-activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an 'emergency brake' that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 microM) and iberiotoxin (100 nM) increase the frequency, but not the amplitude, of spontaneously occurring action potential-dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch-clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired-pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired-pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones.

  5. Role of calcium and potassium channels in effects of hydrogen sulfide on frog myocardial contractility.

    PubMed

    Sitdikova, G F; Khaertdinov, N N; Zefirov, A L

    2011-06-01

    The effects of sodium hydrosulfide NaHS, a donor of hydrogen sulfide H2S, on the force of muscle contraction were examined on isolated myocardial strips from frog ventricles. NaHS decreased the amplitude of muscle contractions in a dose-dependent manner under normal conditions and during inhibition of Ca channels with nifedipine. In contrast, under conditions of blockade of ATP-dependent potassium channels with glibenclamide, NaHS exerted a positive inotropic effect from the first minute of application. Neither blockade, nor activation of ATP-dependent K-channels with glibenclamide modulated the negative inotropic effect of NaHS. Inhibition of K-channels with tetraethylammonium (TEA) (3, 5, 10 mM) or 4-aminopyridine increased the amplitude of myocardial contractions. Preliminary application of 4-aminopyridine or TEA (3 mM) did not eliminate NaHS-induced negative inotropic effect, although higher TEA concentrations (5 or 10 mM) prevented it. The data indicate that the targets of H(2)S in frog myocardium are ATP-dependent, Ca-activated, and voltage-dependent K-channels.

  6. Lipid-dependent gating of a voltage-gated potassium channel

    PubMed Central

    Zheng, Hui; Liu, Weiran; Anderson, Lingyan Y.; Jiang, Qiu-Xing

    2011-01-01

    Recent studies hypothesized that phospholipids stabilize two voltage-sensing arginine residues of certain voltage-gated potassium channels in activated conformations. It remains unclear how lipids directly affect these channels. Here, by examining the conformations of the KvAP in different lipids, we showed that without voltage change, the voltage-sensor domains switched from the activated to the resting state when their surrounding lipids were changed from phospholipids to nonphospholipids. Such lipid-determined conformational change was coupled to the ion-conducting pore, suggesting that parallel to voltage gating, the channel is gated by its annular lipids. Our measurements recognized that the energetic cost of lipid-dependent gating approaches that of voltage gating, but kinetically it appears much slower. Our data support that a channel and its surrounding lipids together constitute a functional unit, and natural nonphospholipids such as cholesterol should exert strong effects on voltage-gated channels. Our first observation of lipid-dependent gating may have general implications to other membrane proteins. PMID:21427721

  7. Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury

    PubMed Central

    Calvo, Margarita; Richards, Natalie; Schmid, Annina B; Barroso, Alejandro; Zhu, Lan; Ivulic, Dinka; Zhu, Ning; Anwandter, Philipp; Bhat, Manzoor A; Court, Felipe A; McMahon, Stephen B; Bennett, David LH

    2016-01-01

    Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. DOI: http://dx.doi.org/10.7554/eLife.12661.001 PMID:27033551

  8. Complex voltage-dependent behavior of single unliganded calcium-sensitive potassium channels.

    PubMed Central

    Talukder, G; Aldrich, R W

    2000-01-01

    study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents. PMID:10653789

  9. Cloning and expression of the human kv4.3 potassium channel.

    PubMed

    Dilks, D; Ling, H P; Cockett, M; Sokol, P; Numann, R

    1999-04-01

    We report on the cloning and expression of hKv4.3, a fast inactivating, transient, A-type potassium channel found in both heart and brain that is 91% homologous to the rat Kv4.3 channel. Two isoforms of hKv4.3 were cloned. One is full length (hKv4.3 long), and the other has a 19 amino acid deletion (hKv4.3 short). RT-PCR shows that the brain contains both forms of the channel RNA, whereas the heart predominantly has the longer version. Both versions of the channel were expressed in Xenopus oocytes, and both contain a significant window or noninactivating current seen near potentials of -30 to -40 mV. The inactivation curve for hKv4.3 short is shifted 10 mV positive relative to hKv4.3 long. This causes the peak window current for the short version to occur near -30 mV and the peak for the longer version to be at -40 mV. There was little difference in the recovery from inactivation or in the kinetics of inactivation between the two isoforms of the channel.

  10. Electrophysiology and pharmacology of tandem domain potassium channel TREK-1 related BDNF synthesis in rat astrocytes.

    PubMed

    Lu, Li; Wang, Weiping; Peng, Ying; Li, Jiang; Wang, Ling; Wang, Xiaoliang

    2014-04-01

    In the present study, the functional properties and pharmacology of two-pore domain potassium channel (K2P) TREK-1 in primary cultured rat brain astrocytes were investigated. Western blot, patch clamping techniques, and ELISA were used to detect the distribution and function of TREK-1 as well as the expression of brain-derived neurotrophic factor (BDNF) on the primary cultured astrocytes. It was shown that TREK-1 protein expressed in astrocytes was 2.4-fold higher than it was expressed in microglia. Single channel recording via patch clamping showed that the TREK-1 outward currents in astrocytes could be activated by arachidonic acid (AA) or chloroform with the conductance of 113 ± 14 and 120 ± 13 pS, respectively. The current was also sensitive to mechanical stretch and intracellular acidification. Negative pressure (-30 cm H2O) and acidification of intracellular solution (pH 6.8 or 6.3) both enhanced TREK-1 channel open probability significantly. Further pharmacological studies showed that TREK-1 antagonist penfluridol inhibited AA-induced currents, and both penfluridol and methionine (TREK-1 blockers) significantly increased BDNF level in astrocytes by 50 %. These results indicated that TREK-1 channel current was a major component of K2P currents in astrocytes. TREK-1 channels might play important roles in regulating the function of astrocytes and might be used as a drug target for neuroprotection.

  11. Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    PubMed Central

    Hsu, Po-Hao; Ma, Yu-Ting; Fang, Ya-Ching; Huang, Jing-Jia; Gan, Yu-Ling; Chang, Pei-Tzu; Jow, Guey-Mei; Tang, Chih-Yung; Jeng, Chung-Jiuan

    2017-01-01

    Mammalian Eag1 (Kv10.1) potassium (K+) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K+ channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels. PMID:28098200

  12. Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex

    PubMed Central

    Watson, Helen R.; Wunderley, Lydia; Andreou, Tereza; Warwicker, Jim; High, Stephen

    2013-01-01

    The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. A prolonged association between TMs of the potassium channel subunit, TASK-1 [TWIK (tandem-pore weak inwardly rectifying potassium channel)-related acid-sensitive potassium channel 1], and the Sec61 complex suggests that the ER translocon co-ordinates the folding/assembly of the TMs present in the nascent chain. The N-terminus of both TASK-1 and Kcv (potassium channel protein of chlorella virus), another potassium channel subunit of viral origin, has access to the N-glycosylation machinery located in the ER lumen, indicating that the Sec61 complex can accommodate multiple arrangements/orientations of TMs within the nascent chain, both in vitro and in vivo. Hence the ER translocon can provide the ribosome-bound nascent chain with a dynamic environment in which it can explore a range of different conformations en route to its correct transmembrane topology and final native structure. PMID:24015703

  13. Domain reorientation and rotation of an intracellular assembly regulate conduction in Kir potassium channels.

    PubMed

    Clarke, Oliver B; Caputo, Alessandro T; Hill, Adam P; Vandenberg, Jamie I; Smith, Brian J; Gulbis, Jacqueline M

    2010-06-11

    Potassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.

  14. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    PubMed

    Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

    2014-01-01

    Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  15. The potassium current carried by TREK-1 channels in rat cardiac ventricular muscle.

    PubMed

    Bodnár, Mandy; Schlichthörl, Günter; Daut, Jürgen

    2015-05-01

    We studied the potassium current flowing through TREK-1 channels in rat cardiac ventricular myocytes. We separated the TREK-1 current from other current components by blocking most other channels with a blocker cocktail. We tried to inhibit the TREK-1 current by activating protein kinase A (PKA) with a mixture of forskolin and isobutyl-methylxanthine (IBMX). Activation of PKA blocked an outwardly rectifying current component at membrane potentials positive to -40 mV. At 37 °C, application of forskolin plus IBMX reduced the steady-state outward current measured at positive voltages by about 52 %. Application of the potassium channel blockers quinidine or tetrahexylammonium also reduced the steady-state outward current by about 50 %. Taken together, our results suggest that the increase in temperature from 22 to 37 °C increased the TREK-1 current by a factor of at least 5 and that the average density of the TREK-1 current in rat cardiomyocytes at 37 °C is about 1.5 pA/pF at +30 mV. The contribution of TREK-1 to the action potential was assessed by using a dynamic patch clamp technique. After subtraction of simulated TREK-1 currents, action potential duration at 50 or 90 % repolarisation was increased by about 12 %, indicating that TREK-1 may be functionally important in rat ventricular muscle. During sympathetic stimulation, inhibition of TREK-1 channels via PKA is expected to prolong the action potential primarily in subendocardial myocytes; this may decrease the transmural dispersion of repolarisation and thus may serve to prevent the occurrence of arrhythmias.

  16. Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury

    PubMed Central

    Yilmaz, Tonguç Utku; Yazihan, Nuray; Dalgic, Aydın; Kaya, Ezgi Ermis; Salman, Bulent; Kocak, Mehtap; Akcil, Ethem

    2015-01-01

    Background & objectives: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions. Methods: Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by Western blot. Results: Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (P<0.01) whereas glibenclamide decreased (P<0.05) HIF-1 α mRNA expression. Glibenclamide significantly (P<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group. Interpretation & conclusions: Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic

  17. Ion Concentration- and Voltage-Dependent Push and Pull Mechanisms of Potassium Channel Ion Conduction.

    PubMed

    Kasahara, Kota; Shirota, Matsuyuki; Kinoshita, Kengo

    2016-01-01

    The mechanism of ion conduction by potassium channels is one of the central issues in physiology. In particular, it is still unclear how the ion concentration and the membrane voltage drive ion conduction. We have investigated the dynamics of the ion conduction processes in the Kv1.2 pore domain, by molecular dynamics (MD) simulations with several different voltages and ion concentrations. By focusing on the detailed ion movements through the pore including selectivity filter (SF) and cavity, we found two major conduction mechanisms, called the III-IV-III and III-II-III mechanisms, and the balance between the ion concentration and the voltage determines the mechanism preference. In the III-IV-III mechanism, the outermost ion in the pore is pushed out by a new ion coming from the intracellular fluid, and four-ion states were transiently observed. In the III-II-III mechanism, the outermost ion is pulled out first, without pushing by incoming ions. Increases in the ion concentration and voltage accelerated ion conductions, but their mechanisms were different. The increase in the ion concentrations facilitated the III-IV-III conductions, while the higher voltages increased the III-II-III conductions, indicating that the pore domain of potassium channels permeates ions by using two different driving forces: a push by intracellular ions and a pull by voltage.

  18. Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel

    PubMed Central

    Nozaki, Tomohiro; Ozawa, Shin-ichiro; Harada, Hitomi; Kimura, Tomomi; Osawa, Masanori; Shimada, Ichio

    2016-01-01

    Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD. PMID:27853286

  19. Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.

    PubMed

    Waters, Christopher W; Varuzhanyan, Grigor; Talmadge, Robert J; Voss, Andrew A

    2013-05-28

    Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

  20. The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography.

    PubMed

    Kuang, Qie; Purhonen, Pasi; Jegerschöld, Caroline; Hebert, Hans

    2014-01-01

    The kch gene, the only potassium channel gene in Escherichia coli, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6Å resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.

  1. Antisense suppression of potassium channel expression demonstrates its role in maturation of the action potential.

    PubMed

    Vincent, A; Lautermilch, N J; Spitzer, N C

    2000-08-15

    A developmental increase in delayed rectifier potassium current (I(Kv)) in embryonic Xenopus spinal neurons is critical for the maturation of excitability and action potential waveform. Identifying potassium channel genes that generate I(Kv) is essential to understanding the mechanisms by which they are controlled. Several Kv genes are upregulated during embryogenesis in parallel with increases in I(Kv) and produce delayed rectifier current when heterologously expressed, indicating that they could encode channels underlying this current. We used antisense (AS) cRNA to test the contribution of xKv3.1 to the maturation of I(Kv), because xKv3.1 AS appears to suppress specifically heterologous expression of potassium current by xKv3.1 mRNA. The injection of xKv3.1 AS into embryos reduces endogenous levels of xKv3.1 mRNA in the developing spinal cord and reduces the amplitude and rate of activation of I(Kv) in 40% of cultured neurons, similar to the percentage of neurons in which endogenous xKv3.1 transcripts are detected. The current in these mature neurons resembles that at an earlier stage of differentiation before the appearance of xKv3.1 mRNA. Furthermore, AS expression increases the duration of the action potential in 40% of the neurons. No change in voltage-dependent calcium current is observed, suggesting that the decrease in I(Kv) is sufficient to account for lengthening of the action potential. Computer-simulated action potentials incorporating observed reductions in amplitude and rate of activation of I(Kv) exhibit an increase in duration similar to that observed experimentally. Thus xKv3.1 contributes to the maturation of I(Kv) in a substantial percentage of these developing spinal neurons.

  2. Dendritic A-type potassium channel subunit expression in CA1 hippocampal interneurons

    PubMed Central

    Menegola, Milena; Misonou, Hiroaki; Vacher, Helene; Trimmer, James S.

    2008-01-01

    Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and KChIP1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal CA1 neurons. Immunohistochemistry was performed on 40 μm rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with unique dendritic A-type channel complement and ability to control BAPs. PMID:18495361

  3. sigma Receptor activation blocks potassium channels and depresses neuroexcitability in rat intracardiac neurons.

    PubMed

    Zhang, Hongling; Cuevas, Javier

    2005-06-01

    The sigma receptors have been implicated in the regulation of the cardiovascular system, and sigma-1 receptor transcripts have been found in parasympathetic intracardiac neurons. However, the cellular function of sigma-1 receptors in these cells remains to be determined. Effects of sigma receptor activation on voltage-activated K(+) channels and action potential firing were studied in isolated intracardiac neurons using whole-cell patch-clamp recording techniques. Activation of sigma receptors reversibly blocked delayed outwardly rectifying potassium channels, large conductance Ca(2+)-sensitive K(+) channels, and the M-current with maximal inhibition >80%. The inhibition of K(+) channels by sigma ligands was dose-dependent, and the rank order potency of (+)-pentazocine > ibogaine > 1,3-di-O-tolyguanidin (DTG) suggests that the effect is mediated by sigma-1 receptor activation. Preincubation of neurons with the irreversible sigma receptor antagonist metaphit blocked DTG-induced inhibition of K(+) channels, confirming that the effect is mediated by sigma receptor activation. Although bath application of sigma ligands depolarized intracardiac neurons, the number of action potentials fired by the cells in response to depolarizing current pulses was decreased in the presence of these drugs. Neither dialysis of the neurons nor application of intracellular 5'-O-(2-thiodiphosphate) trilithium salt inhibited the effect of sigma receptors on K(+) channels, which suggests that the signal transduction pathway does not involve a diffusible cytosolic second messenger or a G protein. Together, these data suggest that sigma-1 receptors are directly coupled to K(+) channels in intracardiac neurons. Furthermore, activation of sigma-1 receptors depresses the excitability of intracardiac neurons and is thus likely to block parasympathetic input to the heart.

  4. Gambierol Inhibition of Voltage-Gated Potassium Channels Augments Spontaneous Ca2+ Oscillations in Cerebrocortical Neurons

    PubMed Central

    Cao, Zhengyu; Cui, Yanjun; Busse, Eric; Mehrotra, Suneet; Rainier, Jon D.

    2014-01-01

    Gambierol is a marine polycyclic ether toxin produced by the marine dinoflagellate Gambierdiscus toxicus and is a member of the ciguatoxin toxin family. Gambierol has been demonstrated to be either a low-efficacy partial agonist/antagonist of voltage-gated sodium channels or a potent blocker of voltage-gated potassium channels (Kvs). Here we examined the influence of gambierol on intact cerebrocortical neurons. We found that gambierol produced both a concentration-dependent augmentation of spontaneous Ca2+ oscillations, and an inhibition of Kv channel function with similar potencies. In addition, an array of selective as well as universal Kv channel inhibitors mimicked gambierol in augmenting spontaneous Ca2+ oscillations in cerebrocortical neurons. These data are consistent with a gambierol blockade of Kv channels underlying the observed increase in spontaneous Ca2+ oscillation frequency. We also found that gambierol produced a robust stimulation of phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Gambierol-stimulated ERK1/2 activation was dependent on both inotropic [N-methyl-d-aspartate (NMDA)] and type I metabotropic glutamate receptors (mGluRs) inasmuch as MK-801 [NMDA receptor inhibitor; (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], S-(4)-CGP [S-(4)-carboxyphenylglycine], and MTEP [type I mGluR inhibitors; 3-((2-methyl-4-thiazolyl)ethynyl) pyridine] attenuated the response. In addition, 2-aminoethoxydiphenylborane, an inositol 1,4,5-trisphosphate receptor inhibitor, and U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C inhibitor, both suppressed gambierol-induced ERK1/2 activation, further confirming the role of type I mGluR-mediated signaling in the observed ERK1/2 activation. Finally, we found that gambierol produced a concentration-dependent stimulation of neurite outgrowth that was mimicked by 4-aminopyridine, a universal potassium

  5. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

  6. Potassium channels control the interaction between active dendritic integration compartments in layer 5 cortical pyramidal neurons

    PubMed Central

    Harnett, Mark T.; Xu, Ning-Long; Magee, Jeffrey C.; Williams, Stephen R.

    2013-01-01

    Active dendritic synaptic integration enhances the computational power of neurons. Such nonlinear processing generates an object-localization signal in the apical dendritic tuft of layer 5B cortical pyramidal neurons during sensory-motor behaviour. Here we employ electrophysiological and optical approaches in brain-slices and behaving animals to investigate how excitatory synaptic input to this distal dendritic compartment influences neuronal output. We find that active dendritic integration throughout the apical dendritic tuft is highly compartmentalized by voltage-gated potassium (KV) channels. A high-density of both transient and sustained KV channels was observed in all apical dendritic compartments. These channels potently regulated the interaction between apical dendritic tuft, trunk, and axo-somatic integration zones to control neuronal output in vitro as well as the engagement of dendritic nonlinear processing in vivo during sensory-motor behaviour. Thus, KV channels dynamically tune the interaction between active dendritic integration compartments in layer 5B pyramidal neurons to shape behaviourally relevant neuronal computations. PMID:23931999

  7. Differential expression of two-pore domain potassium channels in rat cerebellar granule neurons.

    PubMed

    Burgos, Paulina; Zúñiga, Rafael; Domínguez, Pedro; Delgado-López, Fernando; Plant, Leigh D; Zúñiga, Leandro

    2014-10-31

    Two pore domain potassium (K2P) channels are mostly present in the central nervous system (CNS) where they play important roles in modulating neuronal excitability. K2P channels give rise to background K(+) currents (IKSO) a key component in setting and maintaining the resting membrane potential in excitable cells. Here, we studied the expression and relative abundances of K2P channels in cerebellar granule neurons (CGNs), combining molecular biology, electrophysiology and immunologic techniques. The CGN IKSO was very sensitive to external pH, as previously reported. Quantitative determination of mRNA expression level demonstrated the existence of an accumulation pattern of transcripts in CGN that encode K2P9>K2P1>K2P3>K2P18>K2P2=K2P10>K2P4>K2P5 subunits. The presence of the major K2P subunits expressed was then confirmed by Western blot and immunofluorescence analysis, demonstrating robust expression of K2P1 (TWIK-1), K2P3 (TASK-1), K2P9 (TASK-3) and K2P18 (TRESK) channel protein. Based, on these results, it is concluded that K2P1, -3, -9 and -18 subunits represent the majority component of IKSO current in CGN.

  8. Mechanism and Energetics of Charybdotoxin Unbinding from a Potassium Channel from Molecular Dynamics Simulations

    PubMed Central

    Chen, Po-chia; Kuyucak, Serdar

    2009-01-01

    Ion channel-toxin complexes are ideal systems for computational studies of protein-ligand interactions, because, in most cases, the channel axis provides a natural reaction coordinate for unbinding of a ligand and a wealth of physiological data is available to check the computational results. We use a recently determined structure of a potassium channel-charybdotoxin complex in molecular dynamics simulations to investigate the mechanism and energetics of unbinding. Pairs of residues on the channel protein and charybdotoxin that are involved in the binding are identified, and their behavior is traced during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force for the unbinding of charybdotoxin is constructed from the umbrella sampling simulations using the weighted histogram analysis method, and barriers observed are correlated with specific breaking of interactions and influx of water molecules into the binding site. Charybdotoxin is found to undergo conformational changes as a result of the reaction coordinate choice—a nontrivial decision for larger ligands—which we explore in detail, and for which we propose solutions. Agreement between the calculated and the experimental binding energies is obtained once the energetic consequences of these conformational changes are included in the calculations. PMID:19348743

  9. Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel.

    PubMed

    Schow, Eric V; Freites, J Alfredo; Nizkorodov, Alex; White, Stephen H; Tobias, Douglas J

    2012-07-01

    Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.

  10. Membrane lipids tune synaptic transmission by direct modulation of presynaptic potassium channels.

    PubMed

    Carta, Mario; Lanore, Frederic; Rebola, Nelson; Szabo, Zsolt; Da Silva, Silvia Viana; Lourenço, Joana; Verraes, Agathe; Nadler, André; Schultz, Carsten; Blanchet, Christophe; Mulle, Christophe

    2014-02-19

    Voltage-gated potassium (Kv) channels are involved in action potential (AP) repolarization in excitable cells. Exogenous application of membrane-derived lipids, such as arachidonic acid (AA), regulates the gating of Kv channels. Whether membrane-derived lipids released under physiological conditions have an impact on neuronal coding through this mechanism is unknown. We show that AA released in an activity-dependent manner from postsynaptic hippocampal CA3 pyramidal cells acts as retrograde messenger, inducing a robust facilitation of mossy fiber (Mf) synaptic transmission over several minutes. AA acts by broadening presynaptic APs through the direct modulation of Kv channels. This form of short-term plasticity can be triggered when postsynaptic cell fires with physiologically relevant patterns and sets the threshold for the induction of the presynaptic form of long-term potentiation (LTP) at hippocampal Mf synapses. Hence, direct modulation of presynaptic Kv channels by activity-dependent release of lipids serves as a physiological mechanism for tuning synaptic transmission.

  11. Channeling your inner ear potassium: K(+) channels in vestibular hair cells.

    PubMed

    Meredith, Frances L; Rennie, Katherine J

    2016-08-01

    During development of vestibular hair cells, K(+) conductances are acquired in a specific pattern. Functionally mature vestibular hair cells express different complements of K(+) channels which uniquely shape the hair cell receptor potential and filtering properties. In amniote species, type I hair cells (HCI) have a large input conductance due to a ubiquitous low-voltage-activated K(+) current that activates with slow sigmoidal kinetics at voltages negative to the membrane resting potential. In contrast type II hair cells (HCII) from mammalian and non-mammalian species have voltage-dependent outward K(+) currents that activate rapidly at or above the resting membrane potential and show significant inactivation. A-type, delayed rectifier and calcium-activated K(+) channels contribute to the outward K(+) conductance and are present in varying proportions in HCII. In many species, K(+) currents in HCII in peripheral locations of vestibular epithelia inactivate more than HCII in more central locations. Two types of inward rectifier currents have been described in both HCI and HCII. A rapidly activating K(+)-selective inward rectifier current (IK1, mediated by Kir2.1 channels) predominates in HCII in peripheral zones, whereas a slower mixed cation inward rectifier current (Ih), shows greater expression in HCII in central zones of vestibular epithelia. The implications for sensory coding of vestibular signals by different types of hair cells are discussed. This article is part of a Special Issue entitled .

  12. Conformational dynamics of the inner pore helix of voltage-gated potassium channels

    NASA Astrophysics Data System (ADS)

    Choe, Seungho; Grabe, Michael

    2009-06-01

    Voltage-gated potassium (Kv) channels control the electrical excitability of neurons and muscles. Despite this key role, how these channels open and close or gate is not fully understood. Gating is usually attributed to the bending and straightening of pore-lining helices at glycine and proline residues. In this work we focused on the role of proline in the Pro-Val-Pro (PVP) motif of the inner S6 helix in the Kv1.2 channel. We started by developing a simple hinged-rod model to fully explore the configurational space of bent helices and we related these configurations to the degree of pore opening. We then carried out fully atomistic simulations of the S6 helices and compared these simulations to the hinged-rod model. Both methods suggest that Kv1 channels are not tightly closed when the inner helices are straight, unlike what is seen in the non-PVP containing channels KcsA and KirBac. These results invite the possibility that the S6 helices may be kinked when Kv1 channels are closed. Our simulations indicate that the wild-type helix adopts multiple spatially distinct configurations, which is consistent with its role in adopting a closed state and an open state. The two most dominant configurational basins correspond to a 6 Å movement of the helix tail accompanied by the PVP region undergoing a local α-helix to 310-helix transition. We explored how single point mutations affect the propensity of the S6 helix to adopt particular configurations. Interestingly, mutating the first proline, P405 (P473 in Shaker), to alanine completely removed the bistable nature of the S6 helix possibly explaining why this mutation compromises the channel. Next, we considered four other mutations in the area known to affect channel gating and we saw similarly dramatic changes to the helix's dynamics and range of motion. Our results suggest a possible mechanism of helix pore closure and they suggest differences in the closed state of glycine-only channels, like KcsA, and PVP containing

  13. Involvement of a membrane potassium channel in heparan sulphate-induced activation of macrophages.

    PubMed

    Ren, Jian-Dong; Fan, Li; Tian, Fu-Zhou; Fan, Kai-Hua; Yu, Bo-Tao; Jin, Wei-Hua; Tan, Yong-Hong; Cheng, Long

    2014-03-01

    Increasing evidence has demonstrated that Toll-like receptor 4 (TLR4) -mediated systemic inflammatory response syndrome accompanied by multiple organ failure, is one of the most common causes of death in patients with severe acute pancreatitis. Recent reports have revealed that heparan sulphate (HS) proteoglycan, a component of extracellular matrices, potentiates the activation of intracellular pro-inflammatory responses via TLR4, contributing to the aggravation of acute pancreatitis. However, little is known about the participants in the HS/TLR4-mediated inflammatory cascades. Our previous work provided a clue that a membrane potassium channel (MaxiK) is responsible for HS-induced production of inflammatory cytokines. Therefore, in this report we attempted to reveal the roles of MaxiK in the activation of macrophages stimulated by HS. Our results showed that incubation of RAW264.7 cells with HS up-regulated MaxiK and TLR4 expression levels. HS could also activate MaxiK channels to promote the efflux of potassium ions from cells, as measured by the elevated activity of caspase-1, whereas this was significantly abolished by treatment with paxilline, a specific blocker of the MaxiK channel. Moreover, it was found that paxilline substantially inhibited HS-induced activation of several different transcription factors in macrophages, including nuclear factor-κB, p38 and interferon regulatory factor-3, followed by decreased production of tumour necrosis factor-α and interferon-β. Taken together, our investigation provides evidence that the HS/TLR4-mediated intracellular inflammatory cascade depends on the activation of MaxiK, which may offer an important opportunity for a new approach in therapeutic strategies of severe acute pancreatitis.

  14. Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

    PubMed Central

    Zhang, Yan-Hui; Wu, Wei; Chen, Kui-Hao; Liu, Yi; Deng, Chun-Yu; Yu, Xi-Yong; Jin, Man-Wen; Li, Gui-Rong

    2012-01-01

    Allitridi (diallyl trisulfide) is an active compound (volatile oil) from garlic. The previous studies reported that allitridi had anti-arrhythmic effect. The potential ionic mechanisms are, however, not understood. The present study was designed to determine the effects of allitridi on cardiac potassium channels expressed in HEK 293 cells using a whole-cell patch voltage-clamp technique and mutagenesis. It was found that allitridi inhibited hKv4.3 channels (IC50 = 11.4 µM) by binding to the open channel, shifting availability potential to hyperpolarization, and accelerating closed-state inactivation of the channel. The hKv4.3 mutants T366A, T367A, V392A, and I395A showed a reduced response to allitridi with IC50s of 35.5 µM, 44.7 µM, 23.7 µM, and 42.4 µM. In addition, allitridi decreased hKv1.5, hERG, hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with IC50s of 40.2 µM, 19.6 µM and 17.7 µM. However, it slightly inhibited hKir2.1 current (100 µM, inhibited by 9.8% at −120 mV). Our results demonstrate for the first time that allitridi preferably blocks hKv4.3 current by binding to the open channel at T366 and T367 of P-loop helix, and at V392 and I395 of S6 domain. It has a weak inhibition of hKv1.5, hERG, and hKCNQ1/hKCNE1 currents. These effects may account for its anti-arrhythmic effect observed in experimental animal models. PMID:23272117

  15. Distribution of High-Conductance Calcium-Activated Potassium Channels in Rat Vestibular Epithelia

    PubMed Central

    Schweizer, Felix E.; Savin, David; Luu, Cindy; Sultemeier, David R.; Hoffman, Larry F.

    2011-01-01

    Voltage- and calcium-activated potassium channels (BK) are important regulators of neuronal excitability. BK channels seem to be crucial for frequency tuning in nonmammalian vestibular and auditory hair cells. However, there are a paucity of data concerning BK expression in mammalian vestibular hair cells. We therefore investigated the localization of BK channels in mammalian vestibular hair cells, specifically in rat vestibular neuroepithelia. We find that only a subset of hair cells in the utricle and the crista ampullaris express BK channels. BK-positive hair cells are located mainly in the medial striolar region of the utricle, where they constitute at most 12% of hair cells, and in the central zone of the horizontal crista. A majority of BK-positive hair cells are encapsulated by a calretinin-positive calyx defining them as type I cells. The remainder are either type I cells encapsulated by a calretinin-negative calyx or type II hair cells. Surprisingly, the number of BK-positive hair cells in the utricle peaks in juvenile rats and declines in early adulthood. BK channels were not found in vestibular afferent dendrites or somata. Our data indicate that BK channel expression in the mammalian vestibular system differs from the expression pattern in the mammalian auditory and the nonmammalian vestibular system. The molecular diversity of vestibular hair cells indicates a functional diversity that has not yet been fully characterized. The predominance of BK-positive hair cells within the medial striola of juvenile animals suggests that they contribute to a scheme of highly lateralized coding of linear head movements during late development. PMID:19731297

  16. Structure of KCNE1 and implications for how it modulates the KCNQ1 potassium channel.

    PubMed

    Kang, Congbao; Tian, Changlin; Sönnichsen, Frank D; Smith, Jarrod A; Meiler, Jens; George, Alfred L; Vanoye, Carlos G; Kim, Hak Jun; Sanders, Charles R

    2008-08-05

    KCNE1 is a single-span membrane protein that modulates the voltage-gated potassium channel KCNQ1 (K V7.1) by slowing activation and enhancing channel conductance to generate the slow delayed rectifier current ( I Ks) that is critical for the repolarization phase of the cardiac action potential. Perturbation of channel function by inherited mutations in KCNE1 or KCNQ1 results in increased susceptibility to cardiac arrhythmias and sudden death with or without accompanying deafness. Here, we present the three-dimensional structure of KCNE1. The transmembrane domain (TMD) of KCNE1 is a curved alpha-helix and is flanked by intra- and extracellular domains comprised of alpha-helices joined by flexible linkers. Experimentally restrained docking of the KCNE1 TMD to a closed state model of KCNQ1 suggests that KCNE1 slows channel activation by sitting on and restricting the movement of the S4-S5 linker that connects the voltage sensor to the pore domain. We postulate that this is an adhesive interaction that must be disrupted before the channel can be opened in response to membrane depolarization. Docking to open KCNQ1 indicates that the extracellular end of the KCNE1 TMD forms an interface with an intersubunit cleft in the channel that is associated with most known gain-of-function disease mutations. Binding of KCNE1 to this "gain-of-function cleft" may explain how it increases conductance and stabilizes the open state. These working models for the KCNE1-KCNQ1 complexes may be used to formulate testable hypotheses for the molecular bases of disease phenotypes associated with the dozens of known inherited mutations in KCNE1 and KCNQ1.

  17. Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius).

    PubMed

    Mamidala, Praveen; Mittapelly, Priyanka; Jones, Susan C; Piermarini, Peter M; Mittapalli, Omprakash

    2013-04-01

    The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect 'Kir3' clade, which we designate as 'ClKir3'. The other was a partial cDNA that encodes a protein with similarity to both the insect 'Kir1' and 'Kir2' clades, which we designate as 'ClKir1/2'. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology.

  18. Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels.

    PubMed

    Zhang, Yalan; Brown, Maile R; Hyland, Callen; Chen, Yi; Kronengold, Jack; Fleming, Matthew R; Kohn, Andrea B; Moroz, Leonid L; Kaczmarek, Leonard K

    2012-10-31

    Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na(+)-activated K(+) channels (K(Na)), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.

  19. Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels.

    PubMed

    Baronas, Victoria A; Yang, Runying; Vilin, Yury Y; Kurata, Harley T

    2016-01-01

    Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.

  20. Regulation of human cardiac potassium channels by full-length KCNE3 and KCNE4

    PubMed Central

    Abbott, Geoffrey W.

    2016-01-01

    Voltage-gated potassium (Kv) channels comprise pore-forming α subunits and a multiplicity of regulatory proteins, including the cardiac-expressed and cardiac arrhythmia-linked transmembrane KCNE subunits. After recently uncovering novel, N-terminally extended (L) KCNE3 and KCNE4 isoforms and detecting their transcripts in human atrium, reported here are their functional effects on human cardiac Kv channel α subunits expressed in Xenopus laevis oocytes. As previously reported for short isoforms KCNE3S and KCNE4S, KCNE3L inhibited hERG; KCNE4L inhibited Kv1.1; neither form regulated the HCN1 pacemaker channel. Unlike KCNE4S, KCNE4L was a potent inhibitor of Kv4.2 and Kv4.3; co-expression of cytosolic β subunit KChIP2, which regulates Kv4 channels in cardiac myocytes, partially relieved Kv4.3 but not Kv4.2 inhibition. Inhibition of Kv4.2 and Kv4.3 by KCNE3L was weaker, and its inhibition of Kv4.2 abolished by KChIP2. KCNE3L and KCNE4L also exhibited subunit-specific effects on Kv4 channel complex inactivation kinetics, voltage dependence and recovery. Further supporting the potential physiological significance of the robust functional effects of KCNE4L on Kv4 channels, KCNE4L protein was detected in human atrium, where it co-localized with Kv4.3. The findings establish functional effects of novel human cardiac-expressed KCNE isoforms and further contribute to our understanding of the potential mechanisms influencing cardiomyocyte repolarization. PMID:27922120

  1. Missense mutations in the sodium-gated potassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy.

    PubMed

    Heron, Sarah E; Smith, Katherine R; Bahlo, Melanie; Nobili, Lino; Kahana, Esther; Licchetta, Laura; Oliver, Karen L; Mazarib, Aziz; Afawi, Zaid; Korczyn, Amos; Plazzi, Giuseppe; Petrou, Steven; Berkovic, Samuel F; Scheffer, Ingrid E; Dibbens, Leanne M

    2012-11-01

    We performed genomic mapping of a family with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and intellectual and psychiatric problems, identifying a disease-associated region on chromosome 9q34.3. Whole-exome sequencing identified a mutation in KCNT1, encoding a sodium-gated potassium channel subunit. KCNT1 mutations were identified in two additional families and a sporadic case with severe ADNFLE and psychiatric features. These findings implicate the sodium-gated potassium channel complex in ADNFLE and, more broadly, in the pathogenesis of focal epilepsies.

  2. Three-dimensional structure of kappa-conotoxin PVIIA, a novel potassium channel-blocking toxin from cone snails.

    PubMed

    Savarin, P; Guenneugues, M; Gilquin, B; Lamthanh, H; Gasparini, S; Zinn-Justin, S; Ménez, A

    1998-04-21

    kappa-Conotoxin PVIIA from the venom of Conus purpurascens is the first cone snail toxin that was described to block potassium channels. We synthesized chemically this toxin and showed that its disulfide bridge pattern is similar to those of omega- and delta-conotoxins. kappa-conotoxin competes with radioactive alpha-dendrotoxin for binding to rat brain synaptosomes, confirming its capacity to bind to potassium channels; however, it behaves as a weak competitor. The three-dimensional structure of kappa-conotoxin PVIIA, as elucidated by NMR spectroscopy and molecular modeling, comprises two large parallel loops stabilized by a triple-stranded antiparallel beta-sheet and three disulfide bridges. The overall fold of kappa-conotoxin is similar to that of calcium channel-blocking omega-conotoxins but differs from those of potassium channel-blocking toxins from sea anemones, scorpions, and snakes. Local topographies of kappa-conotoxin PVIIA that might account for its capacity to recognize Kv1-type potassium channels are discussed.

  3. Targeting Ion Channels: An Important Therapeutic Implication in Gastrointestinal Dysmotility in Patients With Spinal Cord Injury

    PubMed Central

    Radulovic, Miroslav; Anand, Preeti; Korsten, Mark A; Gong, Bing

    2015-01-01

    Gastrointestinal (GI) dysmotility is a severe, and common complication in patients with spinal cord injury (SCI). Current therapeutic methods using acetylcholine analogs or laxative agents have unwanted side effects, besides often fail to have desired effect. Various ion channels such as ATP-sensitive potassium (KATP) channel, calcium ions (Ca2+)-activated potassium ions (K+) channels, voltage-sensitive Ca2+ channels and chloride ion (Cl−) channels are abundantly expressed in GI tissues, and play an important role in regulating GI motility. The release of neurotransmitters from the enteric nerve terminal, innervating GI interstitial cells of Cajal (ICC), and smooth muscle cells (SMC), causes inactivation of K+ and Cl− channels, increasing Ca2+ influx into cytoplasm, resulting in membrane depolarization and smooth muscle contraction. Thus, agents directly regulating ion channels activity either in ICC or in SMC may affect GI peristalsis and would be potential therapeutic target for the treatment of GI dysmotility with SCI. PMID:26424038

  4. Angiotensin II inhibits the ROMK-like small conductance K channel in renal cortical collecting duct during dietary potassium restriction.

    PubMed

    Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M; Wang, Wen-Hui

    2007-03-02

    Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

  5. Angiotensin II Inhibits the ROMK-like Small Conductance K Channel in Renal Cortical Collecting Duct during Dietary Potassium Restriction*

    PubMed Central

    Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M.; Wang, Wen-Hui

    2010-01-01

    Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction. PMID:17194699

  6. Electrophysiological consequences of KATP Gain-of-function in the heart: Conduction abnormalities in Cantu Syndrome

    PubMed Central

    Levin, Mark D.; Zhang, Haixia; Uchida, Keita; Grange, Dorothy K.; Singh, Gautam K.; Nichols, Colin G.

    2015-01-01

    Background Gain-of-function (GOF) mutations in the KATP channel subunits Kir6.1 and SUR2 cause Cantu syndrome (CS), a disease characterized by multiple cardiovascular abnormalities. Objective To better understand the electrophysiological consequences of such GOF mutations in the heart. Methods We generated transgenic mice (Kir6.1-GOF) expressing ATP-insensitive Kir6.1[G343D] subunits under α-myosin heavy chain (α-MHC) promoter control, to target gene expression specifically in cardiomyocytes, and carried out patch-clamp experiments on isolated ventricular myocytes, invasive electrophysiology on anesthetized mice. Results In Kir6.1-GOF ventricular myocytes, KATP channels show decreased ATP sensitivity, but there is no significant change in current density. Ambulatory ECG recordings on Kir6.1-GOF mice reveal AV nodal conduction abnormalities and junctional rhythm. Invasive electrophysiological analyses reveal slowing of conduction and conduction failure through the AV node, but no increase in susceptibility to atrial or ventricular ectopic activity. Surface electrocardiograms recorded from CS patients also demonstrate first degree AV block, and fascicular block. Conclusions The primary electrophysiological consequence of cardiac KATP GOF is on the conduction system, particularly the AV node, resulting in conduction abnormalities in CS patients, who carry KATP GOF mutations. PMID:26142302

  7. Effects of fluoxetine on protein expression of potassium ion channels in the brain of chronic mild stress rats

    PubMed Central

    Chen, Chunlin; Wang, Ling; Rong, Xianfang; Wang, Weiping; Wang, Xiaoliang

    2014-01-01

    The purpose of this study is to investigate the expression of major potassium channel subtypes in the brain of chronical mild stress (CMS) rats and reveal the effects of fluoxetine on the expression of these channels. Rats were exposed to a variety of unpredictable stress for three weeks and induced anhedonia, lower sucrose preference, locomotor activity and lower body weight. The protein expressions were determined by Western blot. CMS significantly increased the expression of Kv2.1