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The KCNE2 single transmembrane-spanning voltage-gated potassium (Kv) channel β subunit is ubiquitously expressed and essential for normal function of a variety of cell types, often via regulation of the KCNQ1 Kv channel. A polymorphism upstream of KCNE2 is associated with reduced lung function in human populations, but the pulmonary consequences of KCNE2 gene disruption are unknown. Here, germline deletion of mouse Kcne2 reduced pulmonary expression of potassium channel α subunits Kcnq1 and Kcnb1 but did not alter expression of other Kcne genes. Kcne2 colocalized and coimmunoprecipitated with Kcnq1 in mouse lungs, suggesting the formation of pulmonary Kcnq1-Kcne2 potassium channel complexes. Kcne2 deletion reduced blood O2, increased CO2, increased pulmonary apoptosis, and increased inflammatory mediators TNF-α, IL-6, and leukocytes in bronchoalveolar lavage (BAL) fluids. Consistent with increased pulmonary vascular leakage, Kcne2 deletion increased plasma, BAL albumin, and the BAL:plasma albumin concentration ratio. Kcne2-/- mouse lungs exhibited baseline induction of the reperfusion injury salvage kinase pathway but were less able to respond via this pathway to imposed pulmonary ischemia/reperfusion injury (IRI). We conclude that KCNE2 regulates KCNQ1 in the lungs and is required for normal lung function and resistance to pulmonary IRI. Our data support a causal relationship between KCNE2 gene disruption and lung dysfunction.-Zhou, L., Köhncke, C., Hu, Z., Roepke, T. K., Abbott, G. W. The KCNE2 potassium channel β subunit is required for normal lung function and resilience to ischemia and reperfusion injury.

Many hippocampal CA1 pyramidal cells function as place cells, increasing their firing rate when a specific place field is traversed. The dependence of CA1 place cell firing on position within the place field is asymmetric. We investigated the source of this asymmetry by injecting triangular depolarizing current ramps to approximate the spatially tuned, temporally diffuse depolarizing synaptic input received by these neurons while traversing a place field. Ramps were applied to CA1 pyramidal neurons from male rats in vitro (slice electrophysiology) and in silico (multicompartmental NEURON model). Under control conditions, CA1 neurons fired more action potentials at higher frequencies on the up-ramp versus the down-ramp. This effect was more pronounced for dendritic compared with somatic ramps. We incorporated a four-state Markov scheme for NaV1.6 channels into our model and calibrated the spatial dependence of long-term inactivation according to the literature; this spatial dependence was sufficient to explain the difference in dendritic versus somatic ramps. Long-term inactivation reduced the firing frequency by decreasing open-state occupancy, and reduced spike amplitude during trains by decreasing occupancy in the closed state, which comprises the available pool. PKC activator phorbol-dibutyrate, known to reduce NaV long-term inactivation, removed spike amplitude attenuation in vitro more visibly in dendrites and greatly reduced adaptation, consistent with our hypothesized mechanism. Intracellular application of a peptide inducing long-term NaV inactivation elicited spike amplitude attenuation during spike trains in the soma and greatly enhanced adaptation. Our synergistic experimental/computational approach shows that long-term inactivation of NaV1.6 is a key mechanism of adaptation in CA1 pyramidal cells. [ABSTRACT FROM AUTHOR]

BackgroundHypercholesterolemia‐induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K+ (Kir) channels and that Kir2.1 is an upstream mediator of flow‐induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia‐induced inhibition of flow‐induced NO production and flow‐induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions. Methods and ResultsKir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated–low‐density lipoprotein or isolated from apolipoprotein E–deficient (Apoe−/−) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe−/−mice, Kir2.1+/−/Apoe−/− exhibit the same blunted FIV and flow‐induced NO response as Apoe−/−or Kir2.1+/− alone, but while FIV in Apoe−/− mice can be rescued by cholesterol depletion, in Kir2.1+/−/Apoe−/− mice cholesterol depletion has no effect on FIV. Endothelial‐specific overexpression of Kir2.1 in arteries from Apoe−/− and Kir2.1+/−/Apoe−/− mice results in full rescue of FIV and NO production in Apoe−/− mice with and without the addition of a high‐fat diet. Conversely, endothelial‐specific expression of dominant‐negative Kir2.1 results in the opposite effect. Kir2.1+/−/Apoe−/−mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta. ConclusionsWe conclude that hypercholesterolemia‐induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow‐induced NO production, whereas the stages downstream of flow‐induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

As we previously demonstrated the role of different K+ channels in the action of nicorandil on human saphenous vein (HSV) and human internal mammary artery (HIMA), this study aimed to analyse the contribution of the cGMP pathway in nicorandil-induced vasorelaxation and to determine the involvement of cGMP in the K+ channel-activating effect of nicorandil. An inhibitor of soluble guanylate cyclase (GC), ODQ, significantly inhibited nicorandil-induced relaxation, while ODQ plus glibenclamide, a selective ATP-sensitive K+ (KATP) channel inhibitor, produced a further inhibition of both vessels. In HSV, ODQ in combination with 4-aminopyridine, a blocker of voltage-gated K+ (KV) channels, did not modify the concentration-response to nicorandil compared with ODQ, whereas in HIMA, ODQ plus iberiotoxin, a selective blocker of large-conductance Ca2+-activated K+ (BKCa) channels, produced greater inhibition than ODQ alone. We showed that the cGMP pathway plays a significant role in the vasorelaxant effect of nicorandil on HSV and HIMA. It seems that nicorandil directly opens KATP channels in both vessels and BKCa channels in HIMA, although it is possible that stimulation of GC contributes to KATP channels activation in HIMA. Contrary, the activation of KV channels in HSV is probably due to GC activation and increased levels of cGMP.

Connexin 43 (Cx43) is a gap junction protein expressed in various tissues and organs of vertebrates. Besides functioning as a gap junction, Cx43 also regulates diverse cellular processes like cell growth and differentiation, cell migration, cell survival, etc. Cx43 is critical for normal cardiac functioning and is therefore abundantly expressed in cardiomyocytes. On the other hand, ATP-sensitive potassium (K) channels are metabolic sensors converting metabolic changes into electrical activity. These channels are important in maintaining the neurotransmitter release, smooth muscle relaxation, cardiac action potential repolarization, normal physiology of cellular repolarization, insulin secretion and immune function. Cx43 and K channels are part of the same signaling pathway, regulating cell survival during stress conditions and ischemia/hypoxia preconditioning. However, the underlying molecular mechanism for their combined role in ischemia/hypoxia preconditioning is largely unknown. The current review focuses on understanding the molecular mechanism responsible for the coordinated role of Cx43 and K channel protein in protecting cardiomyocytes against ischemia/hypoxia stress. [ABSTRACT FROM AUTHOR]

Purpose: The present study investigated the putative mechanisms underlying effects of K channel on high glucose (HG)-induced mesangial cell proliferation and tissue inhibitors of metalloproteinases (TIMP)-2 and Collagen IV production. Methods: Rat mesangial cells were subjected to whole cell patch clamp to record the K channel currents under high glucose (HG, 30 mM) condition. Cell proliferation was measured using a CCK-8 assay. The production of TIMP-2 and Collagen IV and AMP-activated protein kinase (AMPK)-signaling pathway activity was assessed by ELISA and Western blotting, respectively. AMPK agonist (AICAR) was used to analyze the role of this kinase. The expression of K subunit (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B) was examined using quantitative real-time PCR (RT-PCR). Results: We found that HG was significant decreases in the expression of Kir6.1, SUB2A and SUB2B, three subunits of K, TIMP-2 production, K channel activity and AMPK activity, while it promoted the cell proliferation and Collagen IV production in rat mesangial cells. Pretreatment with K selective opener (diazoxide, DZX) significantly inhibited HG-induced mesangial cell proliferation, Collagen IV production and decrease in K channel activity in rat mesangial cells, which were reversed by pretreatment of 5-hydroxydecanoate, a selective inhibitor of K. Moreover, AICAR pretreatment inhibited HG-induced decrease in K channel activity. Conclusions: Taken together, activating AMPK-K signaling may protect against HG-induced mesangial cell proliferation and Collagen IV production, and, thereby, provides new insights into the molecular mechanisms underlying early diabetic nephropathy (DN). [ABSTRACT FROM AUTHOR]

Background: To review the distribution and function of K channels, describe the use of K channels openers in clinical trials and make the case that these channels may play a role in headache and migraine. Discussion: K channels are widely present in the trigeminovascular system and play an important role in the regulation of tone in cerebral and meningeal arteries. Clinical trials using synthetic K channel openers report headache as a prevalent-side effect in non-migraine sufferers, indicating that K channel opening may cause headache, possibly due to vascular mechanisms. Whether K channel openers can provoke migraine in migraine sufferers is not known. Conclusion: We suggest that K channels may play an important role in migraine pathogenesis and could be a potential novel therapeutic anti-migraine target. [ABSTRACT FROM AUTHOR]

Key points Large conductance, Ca2+-activated K+ (BKCa) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells., BKCa channel blockers paxilline and iberiotoxin, as well as Ca2+ free solutions and divalent cation Cav channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150 m m K+, single channels had a conductance close to 250 pS, within the range of all known BKCa channels., In current clamped horizontal cells, BKCa channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BKCa channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells., Abstract Large conductance, calcium-activated potassium (BKCa) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca2+] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between −30 and +60 mV is carried primarily in BKCa channels in isolated horizontal cells of rats and mice. Whole-cell outward currents were maximal at +50 mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BKCa channel blocker paxilline (100 n m), iberiotoxin (10 μ m), Ca2+ free solutions and divalent cation Cav channel blockers. It was activated by the BKCa channel activator NS1619 (30 μ m). Single channel recordings revealed the conductance of the channels to be 244 ± 11 pS ( n = 17; symmetrical 150 m m K+) with open probability being both voltage- and Ca2+-dependent. The channels showed fast activation kinetics in response to Ca2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BKCa current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BKCa current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BKCa current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP-1) is an endogenous glucose-lowering hormone and GLP-1 receptor agonists are currently being used as antidiabetic drugs clinically. The canonical signalling pathway (including cAMP, Epac2, protein kinase A (PKA) and K channels) is almost universally accepted as the main mechanism of GLP-1-stimulated insulin secretion. This belief is based on in vitro studies that used nanomolar (1-100 nM) concentrations of GLP-1. Recently, it was found that the physiological concentrations (1-10 pM) of GLP-1 also stimulate insulin secretion from isolated islets, induce membrane depolarization and increase of intracellular [Ca] in isolated β cells/pancreatic islets. These responses were unaffected by PKA inhibitors and occurred without detectable increases in intracellular cAMP and PKA activity. These PKA-independent actions of GLP-1 depend on protein kinase C (PKC), involve activation of the standard GLP-1 receptor (GLP1R) and culminate in activation of phospholipase C (PLC), leading to an elevation of diacylglycerol (DAG), increased L-type Ca and TRPM4/TRPM5 channel activities. Here, we review these recent data and contrast them against the effects of nanomolar concentrations of GLP-1. The differential intracellular signalling activated by low and high concentrations of GLP-1 could provide a clue to explain how GLP-1 exerts different function in the central nervous system and peripheral organs. [ABSTRACT FROM AUTHOR]

Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots ( QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx ( QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo. [ABSTRACT FROM AUTHOR]

BackgroundTwo‐pore K+ channels have emerged as potential targets to selectively regulate cardiac cell membrane excitability; however, lack of specific inhibitors and relevant animal models has impeded the effort to understand the role of 2‐pore K+ channels in the heart and their potential as a therapeutic target. The objective of this study was to determine the role of mechanosensitive 2‐pore K+ channel family member TREK‐1 in control of cardiac excitability. Methods and ResultsCardiac‐specific TREK‐1–deficient mice (αMHC‐Kcnkf/f) were generated and found to have a prevalent sinoatrial phenotype characterized by bradycardia with frequent episodes of sinus pause following stress. Action potential measurements from isolated αMHC‐Kcnk2f/f sinoatrial node cells demonstrated decreased background K+ current and abnormal sinoatrial cell membrane excitability. To identify novel pathways for regulating TREK‐1 activity and sinoatrial node excitability, mice expressing a truncated allele of the TREK‐1–associated cytoskeletal protein βIV‐spectrin (qv4J mice) were analyzed and found to display defects in cell electrophysiology as well as loss of normal TREK‐1 membrane localization. Finally, the βIV‐spectrin/TREK‐1 complex was found to be downregulated in the right atrium from a canine model of sinoatrial node dysfunction and in human cardiac disease. ConclusionsThese findings identify a TREK‐1–dependent pathway essential for normal sinoatrial node cell excitability that serves as a potential target for selectively regulating sinoatrial node cell function.

Soluble epoxide hydrolase (sEH) converts epoxyeicosatrienoic acids that are endothelium-derived hyperpolarizing factors into less active dihydroxyeicosatrienoic acids. Previously, we reported a decrease in adenosine A receptor (AAR) protein levels in sEH knockout (sEH) and an increase in sEH and AAR protein levels in AAR mice. Additionally, K channels are involved in adenosine receptor (AR)-dependent vascular relaxation. Thus, we hypothesize that a potential relationship may exist among sEH over-expression, AAR upregulation, inactivation of K channels, and increased in vascular tone. We performed DMT myograph muscle tension measurements and western blot analysis in isolated mouse mesenteric arteries (MAs) from wild-type (WT) and endothelial over-expression of sEH (Tie2-sEH Tr) mice. Our data revealed that NECA (a non-selective adenosine receptors agonist)-induced relaxation was significantly reduced in Tie2-sEH Tr mice, and CCPA (AAR agonist)-induced contraction was increased in Tie2-sEH Tr mice. AAR-dependent contraction in Tie2-sEH Tr mice was significantly attenuated by pharmacological inhibition of CYP4A (HET0016, 10 µM), PKCα (GO6976, 1 µM), and ERK1/2 (PD58059, 1 µM). Our western blot analysis revealed significantly higher basal protein expression of CYP4A, AAR, and reduced p-ERK in MAs of Tie2-sEH Tr mice. Notably, pinacidil (K channel opener)-induced relaxation was also significantly reduced in MAs of Tie2-sEH Tr mice. Furthermore, K channel-dependent relaxation in MAs was enhanced by inhibition of PKCα and ERK1/2 in WT but not Tie2-sEH Tr mice. In conclusion, our data suggest that over-expression of sEH enhances AAR-dependent contraction and reduces K channel-dependent relaxation in MAs. These results suggest a possible interaction between sEH, AAR, and K channels in regulating vascular tone. [ABSTRACT FROM AUTHOR]

Cardiac K channels are critical determinants of cardiac excitability. In hypertrophied and failing myocardium, alterations in the expression and activity of voltage-gated K currents are frequently observed and contribute to the increased propensity for life-threatening arrhythmias. Thus, understanding the mechanisms of disturbed K channel regulation in heart failure (HF) is of critical importance. Amongst others, Ca/calmodulin-dependent protein kinase II (CaMKII) has been identified as an important regulator of K channel activity. In human HF but also various animal models, increased CaMKII expression and activity has been linked to deteriorated contractile function and arrhythmias. This review will discuss the current knowledge about CaMKII regulation of several K channels, its influence on action potential properties, dispersion of repolarization and arrhythmias with special focus on heart failure.

Aims/hypothesis: Individuals with heterozygous activating mutations of the KCNJ11 gene encoding a subunit of the ATP-sensitive potassium channel (KATP) can usually be treated with oral sulfonylurea (SU) pills in lieu of insulin injections. The aim of this study was to test our hypothesis that younger age at the time of initiation of SU therapy is correlated with lower required doses of SU therapy, shorter transition time and decreased likelihood of requiring additional diabetes medications. Methods: We performed a retrospective cohort study using data on 58 individuals with neonatal diabetes due to KCNJ11 mutations identified through the University of Chicago Monogenic Diabetes Registry (). We assessed the influence of age at initiation of SU therapy on treatment outcomes. Results: HbA fell from an average of 8.5% (69 mmol/mol) before transition to 6.2% (44 mmol/mol) after SU therapy ( p < 0.001). Age of initiation of SU correlated with the dose (mg kg day) of SU required at follow-up ( r = 0.80, p < 0.001). Similar associations were observed across mutation subtypes. Ten participants required additional glucose-lowering medications and all had initiated SU at age 13 years or older. No serious adverse events were reported. Conclusions/interpretation: Earlier age at initiation of SU treatment is associated with improved response to SU therapy. Declining sensitivity to SU may be due to loss of beta cell mass over time in those treated with insulin. Our data support the need for early genetic diagnosis and appropriate personalised treatment in all cases of neonatal diabetes. [ABSTRACT FROM AUTHOR]

This study aims to investigate the signaling mechanism involved in HS-induced modulation of adenosine-mediated vascular tone in the presence or absence of adenosine A receptor (AAR). We hypothesized that HS-induced enhanced vascular relaxation through AAR and epoxyeicosatrienoic acid (EETs) is dependent on peroxisome proliferator-activated receptor gamma (PPARγ) and ATP-sensitive potassium channels (K channels) in AAR mice, while HS-induced vascular contraction to adenosine is dependent on soluble epoxide hydrolase (sEH) that degrades EETs in AAR mice. Organ bath and Western blot techniques were conducted in HS (4 % NaCl) and normal salt (NS, 0.45 % NaCl)-fed AAR and AAR mouse aorta. We found that enhanced vasodilation to AAR agonist, CGS 21680, in HS-fed AAR mice was blocked by PPARγ antagonist (T0070907) and K channel blocker (Glibenclamide). Also, sEH inhibitor (AUDA)-dependent vascular relaxation was mitigated by PPARγ antagonist. PPARγ agonist (Rosiglitazone)-induced relaxation in HS-AAR mice was attenuated by K channel blocker. Conversely, HS-induced contraction in AAR mice was attenuated by sEH inhibitor. Overall, findings from this study that implicates the contribution of EETs, PPARγ and K channels downstream of AAR to mediate enhanced vascular relaxation in response to HS diet while, role of sEH in mediating vascular contraction in HS-fed AAR mice. [ABSTRACT FROM AUTHOR]

The current kinetics of two-pore domain potassium (K) channels resemble those of the steady-state K currents being active during the plateau phase of cardiac action potentials. Recent studies support that K channels contribute to these cardiac currents and thereby influence action potential duration in the heart. Ten of the 15 K channels present in the human genome are sensitive to variations of the extracellular and/or intracellular pH value. This review focuses on a set of K channels which are inhibited by extracellular protons, including the subgroup of tandem of P domains in a weak inward-rectifying K (TWIK)-related acid-sensitive potassium (TASK) and TWIK-related alkaline-activated K (TALK) channels. The role of TWIK-1 in the heart is also discussed since, after successful expression, an extracellular pH dependence, similar to that of TASK-1, was described as a hallmark of TWIK-1. The expression profile in cardiac tissue of different species and the functional data in the heart are summarized. The distinct role of the different acid-sensitive K channels in cardiac electrophysiology, inherited forms of arrhythmias and pharmacology, and their role as drug targets is currently emerging and is the subject of this review. [ABSTRACT FROM AUTHOR]

Two-pore domain potassium (K) channels are implicated in an array of physiological and pathophysiological roles. As a result of their biophysical properties, these channels produce a background leak K current which has a direct effect on cellular membrane potential and activity. The regulation of potassium leak from cells through K channels is of critical importance to cell function, development and survival. Controlling the cell surface expression of these channels is one mode to regulate their function and is achieved through a balance between regulated channel delivery to and retrieval from the cell surface. Here, we explore the modes of retrieval of K channels from the plasma membrane and observe that K channels are endocytosed in both a clathrin-mediated and clathrin-independent manner. K channels use a variety of pathways and show altered internalisation and sorting in response to external cues. These pathways working in concert, equip the cell with a range of approaches to maintain steady state levels of channels and to respond rapidly should changes in channel density be required. [ABSTRACT FROM AUTHOR]

Tandem two-pore potassium channels (Ks) have widespread expression in the central nervous system and periphery where they contribute to background membrane conductance. Some general anaesthetics promote the opening of some of these channels, enhancing potassium currents and thus producing a reduction in neuronal excitability that contributes to the transition to unconsciousness. Similarly, these channels may be recruited during the normal sleep-wake cycle as downstream effectors of wake-promoting neurotransmitters such as noradrenaline, histamine and acetylcholine. These transmitters promote K channel closure and thus an increase in neuronal excitability. Our understanding of the roles of these channels in sleep and anaesthesia has been largely informed by the study of mouse K knockout lines and what is currently predicted by in vitro electrophysiology and channel structure and gating. [ABSTRACT FROM AUTHOR]

Atrial fibrillation and obstructive sleep apnea are responsible for significant morbidity and mortality in the industrialized world. There is a high medical need for novel drugs against both diseases, and here, Kv1.5 channels have emerged as promising drug targets. In humans, TASK-1 has an atrium-specific expression and TASK-1 is also abundantly expressed in the hypoglossal motor nucleus. We asked whether known Kv1.5 channel blockers, effective against atrial fibrillation and/or obstructive sleep apnea, modulate TASK-1 channels. Therefore, we tested Kv1.5 blockers with different chemical structures for their TASK-1 affinity, utilizing two-electrode voltage clamp (TEVC) recordings in Xenopus oocytes. Despite the low structural conservation of Kv1.5 and TASK-1 channels, we found all Kv1.5 blockers analyzed to be even more effective on TASK-1 than on Kv1.5. For instance, the half-maximal inhibitory concentration (IC) values of AVE0118 and AVE1231 (A293) were 10- and 43-fold lower on TASK-1. Also for MSD-D, ICAGEN-4, S20951 (A1899), and S9947, the IC values were 1.4- to 70-fold lower than for Kv1.5. To describe this phenomenon on a molecular level, we used in silico models and identified unexpected structural similarities between the two drug binding sites. Kv1.5 blockers, like AVE0118 and AVE1231, which are promising drugs against atrial fibrillation or obstructive sleep apnea, are in fact potent TASK-1 blockers. Accordingly, block of TASK-1 channels by these compounds might contribute to the clinical effectiveness of these drugs. The higher affinity of these blockers for TASK-1 channels suggests that TASK-1 might be an unrecognized molecular target of Kv1.5 blockers effective in atrial fibrillation or obstructive sleep apnea. [ABSTRACT FROM AUTHOR]

Analysis of published data provided evidence that the main candidates for the role of the end effector for ischemic postconditioning of the heart are: 1) BK-type K channels (big conductance K channels); 2) mitochondrial ATP-sensitive K channels; and 3) MPT pores (mitochondrial permeability transition pores). However, some investigators believe that mitoK channels constitute no more than an intermediate link in the chain of signal events mediating increases in the tolerance of the heart to the actions of ischemia-reperfusion. Of these three structures, MPT pores are the most likely end effector. However, it remains possible that there is no unique molecular complex which is the sole end effector of postconditioning. It may be that there are several effectors mediating the cardioprotective effects of the adaptive postconditioning phenomenon. [ABSTRACT FROM AUTHOR]

Brain ependymal cells, which form an epithelial layer covering the cerebral ventricles, have been shown to play a role in the regulation of cerebrospinal and interstitial fluids. The machinery underlying this, however, remains largely unknown. Here, we report the specific localization of an inwardly rectifying K channel, Kir4.1, on the ependymal cell membrane suggesting involvement of the channel in this function. Immunohistochemical study with confocal microscopy identified Kir4.1 labeling on the lateral but not apical membrane of ependymal cells. Ultrastructural analysis revealed that Kir4.1-immunogold particles were specifically localized and clustered on adjacent membranes at puncta adherens type junctions, whereas an aquaporin water channel, AQP4, that was also detected on the lateral membrane only occurred at components other than adherens junctions. Therefore, in ependymal cells, Kir4.1 and AQP4 are partitioned into distinct membrane compartments that might respectively transport either K or water. Kir4.1 was also expressed in a specialized form of ependymal cell, namely the tanycyte, being abundant in tanycyte processes wrapping neuropils and blood vessels. These specific localizations suggest that Kir4.1 mediates intercellular K exchange between ependymal cells and also K-buffering transport via tanycytes that can interconnect neurons and vessels/ventricles. We propose that ependymal cells and tanycytes differentially operate Kir4.1 and AQP4 actively to control the property of fluids at local areas in the brain. [ABSTRACT FROM AUTHOR]

SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) are potent regulators of several transporters and ion channels. The kinases are under regulation of with-no-K(Lys) (WNK) kinases. The present study explored whether SPAK and/or OSR1 modify the expression and/or activity of the voltage-gated K channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active SPAK, WNK insensitive SPAK, catalytically inactive SPAK, wild-type OSR1, constitutively active OSR1, WNK insensitive OSR1, and catalytically inactive OSR1. Voltage-gated K channel activity was quantified utilizing dual electrode voltage clamp and Kv1.5 channel protein abundance in the cell membrane utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type SPAK and SPAK, but not by SPAK and SPAK. Similarly, Kv1.5 activity and Kv1.5-HA protein abundance were significantly down-regulated by wild-type OSR1 and OSR1, but not by OSR1 and OSR1. Both, SPAK and OSR1 decrease cell membrane Kv1.5 protein abundance and activity. [ABSTRACT FROM AUTHOR]

Closure of ATP-regulated K channels (K channels) plays a central role in glucose-stimulated insulin secretion in beta cells. K channels are also highly expressed in glucagon-producing alpha cells, where their function remains unresolved. Under hypoglycaemic conditions, K channels are open in alpha cells but their activity is low and only ~1% of that in beta cells. Like beta cells, alpha cells respond to hyperglycaemia with K channel closure, membrane depolarisation and stimulation of action potential firing. Yet, hyperglycaemia reciprocally regulates glucagon (inhibition) and insulin secretion (stimulation). Here we discuss how this conundrum can be resolved and how reduced K channel activity, via membrane depolarisation, paradoxically reduces alpha cell Ca entry and glucagon exocytosis. Finally, we consider whether the glucagon secretory defects associated with diabetes can be attributed to impaired K channel regulation and discuss the potential for remedial pharmacological intervention using sulfonylureas. [ABSTRACT FROM AUTHOR]

We have identified a novel splice variant of the human and rat two-pore domain potassium (K) channel TREK-1. The splice variant TREK-1e results from skipping of exon 5, which causes a frame shift in exon 6. The frame shift produces a novel C-terminal amino acid sequence and a premature termination of translation, which leads to a loss of transmembrane domains M3 and M4 and of the second pore domain. RT-PCR experiments revealed a preferential expression of TREK-1e in kidney, adrenal gland, and amygdala. TREK-1e was nonfunctional when expressed in Xenopus oocytes. However, both the surface expression and the current density of full-length TREK-1 were reduced by co-expression of TREK-1e. Live cell imaging in COS-7 cells transfected with GFP-tagged TREK-1e showed that this splice variant was retained in the endoplasmic reticulum (ER). Attachment of the C-terminus of TREK-1e to two different reporter proteins (Kir2.1 and CD8) led to a strong reduction in the surface expression of these fusion proteins. Progressive truncation of the C-terminus of TREK-1e in these reporter constructs revealed a critical region (amino acids 198 to 205) responsible for the intracellular retention. Mutagenesis experiments indicated that amino acids I204 and W205 are key residues mediating the ER retention of TREK-1e. Our results suggest that the TREK-1e splice variant may interfere with the vesicular traffic of full-length TREK-1 channels from the ER to the plasma membrane. Thus, TREK-1e might modulate the copy number of functional TREK-1 channels at the cell surface, providing a novel mechanism for fine tuning of TREK-1 currents. [ABSTRACT FROM AUTHOR]

To perform auditory tasks such as sound localization in the space, auditory neurons in the brain must distinguish sub-millisecond temporal differences in signals from two ears. Such high temporal resolution is possible when each neuron in the ascending auditory pathway fires brief action potential at very accurate timing. Various pre- and postsynaptic machineries ensuring such high temporal precision of auditory synaptic transmission have been identified. Of particular, in this review, the role of K channels in shortening the duration of synaptic potentials will be discussed. First, the contribution of K channels to AP firing of general auditory neurons will be discussed. Then, the focus will be moved to the inner hair cell (IHC)-auditory afferent nerve fiber (ANF) synapses, the first synapses of ascending auditory pathway. Molecular and immunohistological techniques have revealed various K channels in the cell bodies and their processes of ANFs. Since the development of patch-clamp recordings from the ANF dendrites in 2002, it became possible to monitor the IHC-ANF synaptic transmission in greater detail. As revealed in brain auditory synapses, several different K channels appear to participate in reducing the duration of synaptic potentials at the IHC-ANF synapses. In addition, K channels at the ANF dendrites might act as potential targets of efferent feedback from the brain. The hypothesis is that, upon loud sound exposure, efferent neurotransmitters released onto the ANF dendrites activate certain K channels and prevent excitotoxicity of ANFs. Therefore, K channels of the ANF dendrites might provide potential sites of pharmacological actions to prevent noise-induced hearing loss. [ABSTRACT FROM AUTHOR]

Thromboxane A (TXA) is known to stimulate colonic cancer cell proliferation, although the mechanism has not been clarified. In this study, we compared the expression levels of Kv7.1 K channels between human colorectal cancer tissue and the accompanying non-tumor mucosa. Kv7.1 proteins were found to be consistently up-regulated in the cancer tissues from different patients. Kv7.1 was also expressed in human colonic cancer cell lines. Treatment of colonic cancer cells with 9,11-epithio-11,12-methano-thromboxane A (STA), a stable analogue of TXA, significantly increased whole-cell K currents sensitive to chromanol 293B, an inhibitor of Kv7.1 channels, in parallel with an increased expression of Kv7.1 proteins. In contrast, TXB, an inactive metabolite of TXA, had no effects on expression level and function of Kv7.1. A TXA receptor antagonist (SQ29548) and an inhibitor of cAMP-dependent protein kinase (Rp-8-Br-MB-cAMPS) inhibited STA-induced increases in both Kv7.1 expression and chromanol 293B-sensitive K currents. Interestingly, STA-stimulated proliferation of colonic cancer cells was inhibited by chromanol 293B. These results suggest that Kv7.1 channels are involved in the TXA-induced cancer cell proliferation and that they are up-regulated by the TXA receptor-mediated cAMP pathway. [ABSTRACT FROM AUTHOR]

The human KCNK18 gene is predominantly expressed in brain, spinal cord, and dorsal root ganglion neurons. Encoded K18.1K channels are functionally implicated in migraine, pain and anesthesia. Data delineating the in vivo significance of K18.1 are still limited owing to a lack of model systems allowing for rapid, whole organism phenotypic analyses. We hypothesized that zebrafish ( Danio rerio) might close this scientific gap. This work was designed to characterize the zebrafish ortholog of K18.1 in comparison to human K18.1 channels. The complete coding sequence of z KCNK18 was amplified from zebrafish cDNA. Zebrafish KCNK18 expression was assessed by in situ hybridization. Human and zebrafish K18.1 currents were functionally analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. KCNK18 mRNA is expressed in zebrafish brain and eyes. Human and zebrafish K18.1 proteins share 32 % identity. Zebrafish K18.1 channels mediate K-selective background currents that stabilize the negative resting membrane potential. Functional similarities between human and zK18.1 currents include open rectification properties, inhibition by barium, and regulation by signaling molecules protein kinase (PK)C, PKA, and phospholipase C. In contrast to the human ortholog, zK18.1 exhibited reduced sensitivity to elevation of intracellular calcium levels by ionomycin and was virtually insensitive to inhibition by quinidine. Zebrafish and human K18.1 channels share functional and regulatory properties, indicating that the zebrafish may serve as model to assess K18.1 function in vivo. However, distinct differences in K18.1 current regulation require careful consideration when zebrafish data are extrapolated to human physiology. [ABSTRACT FROM AUTHOR]

Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na/H exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K channels. Volume-regulatory K channels include the voltage-gated K channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active JAK2 or inactive JAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K conductance. Coexpression of JAK2 or of JAK2, but not of JAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 μM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 μM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K exit, an effect which may contribute to cell volume regulation. [ABSTRACT FROM AUTHOR]

The effect of potential-dependent potassium uptake at 0-120 mM K on matrix Ca accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K content in the medium was observed due to K uptake into the mitochondria. The accumulation of Ca was shown to depend on K concentration in the medium. At K concentration −30 mM, Ca uptake is decreased due to K-induced membrane depolarization, whereas at higher K concentrations, up to 120 mM K, Ca uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K uptake. Mitochondrial K-channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K uptake as well as K-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca uptake, i.e. increase in Ca uptake at low K content in the medium due to the smaller membrane depolarization and decrease in Ca uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K channel, in the regulation of calcium accumulation in rat brain mitochondria. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Inspired by recent speculation about the potential utility of α-antagonism in the treatment of type 2 diabetes, the study examined the contribution of α-antagonism vs other mechanisms to the antihyperglycaemic activity of the imidazoline (±)-efaroxan. Methods: Effects of the racemate and its pure enantiomers on isolated pancreatic islets and beta cells in vitro, as well as on hyperglycaemia in vivo, were investigated in a comparative manner in mice. Results: In isolated perifused islets, the two enantiomers of efaroxan were equally potent in counteracting inhibition of insulin release by the ATP-dependent K (K) channel-opener diazoxide but (+)-efaroxan, the presumptive carrier of α-antagonistic activity, was by far superior in counteracting inhibition of insulin release by the α-agonist UK14,304. In vivo, (+)-efaroxan improved oral glucose tolerance at 100-fold lower doses than (−)-efaroxan and, in parallel with observations made in vitro, was more effective in counteracting UK14,304-induced than diazoxide-induced hyperglycaemia. The antihyperglycaemic activity of much higher doses of (−)-efaroxan was associated with an opposing pattern (i.e. with stronger counteraction of diazoxide-induced than UK14,304-induced hyperglycaemia), which implicates a different mechanism of action. Conclusions/interpretation: The antihyperglycaemic potency of (±)-efaroxan in mice is almost entirely due to α-antagonism, but high doses can also lower blood glucose via another mechanism. Our findings call for reappraisal of the possible clinical utility of α-antagonistic compounds in recently identified subpopulations of patients in which a congenitally higher level of α-adrenergic activation contributes to the development and pathophysiology of type 2 diabetes. [ABSTRACT FROM AUTHOR]

In order to observe antinociceptive effect of Oxymatrine (OMT) and its effect on voltage-activated K channel, the acetic acid-induced abdominal contraction model of mouse was used to test the antinociceptive effect in vivo, and in vitro, the delayed rectifier K currents (Ik) in PC12 cells (rat pheochromocytoma cells) was recorded using the automated patch-clamp method. The results indicated that after application of OMT, the number of acetic acid-induced animal abdominal contraction was significantly decreased, Ik in PC12 cells was significantly decreased, and showed a concentration-dependent manner. After application of OMT, both the activation and inactivation curves of Ik of PC12 cells were shifted to negative potentials. This study revealed that OMT showed antinociceptive effect in mice. The inhibition of voltage-activated K channel might be one of mechanisms in which the enhanced both activation and inactivation of K channel were involved and might play important roles. [ABSTRACT FROM AUTHOR]

Dronedarone is currently used for the treatment of paroxysmal and persistent atrial fibrillation (AF). Pharmacological inhibition of cardiac two-pore-domain potassium (K) channels results in action potential prolongation and has recently been proposed as novel antiarrhythmic strategy. We hypothesized that blockade of human K channels contributes to the electrophysiological efficacy of dronedarone in AF. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K currents from Xenopus oocytes and Chinese hamster ovary cells. All functional human K channels were screened for dronedarone sensitivity, revealing significant and concentration-dependent inhibition of cardiac K2.1 (TREK1; IC = 26.7 μM) and K3.1 channels (TASK1; IC = 18.7 μM) with maximum current reduction of 60.3 and 65.5 % in oocytes. IC values obtained from mammalian cells yielded 6.1 μM (K2.1) and 5.2 μM (K3.1). The molecular mechanism of action was studied in detail. Dronedarone block affected open and closed channels. K3.1 currents were reduced in frequency-dependent fashion in contrast to K2.1. Mutagenesis studies revealed that amino acid residues implicated in K3.1 drug interactions were not required for dronedarone blockade. The class III antiarrhythmic drug dronedarone targets multiple human cardiac two-pore-domain potassium channels, including atrial-selective K3.1 currents. K current inhibition by dronedarone represents a previously unrecognized mechanism of action that extends the multichannel blocking profile of the drug. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Several glucose-sensing pathways have been implicated in glucose-triggered secretion of glucagon-like peptide-1 (GLP-1) from intestinal L cells. One involves glucose metabolism and closure of ATP-sensitive K channels, and another exploits the electrogenic nature of Na-coupled glucose transporters (SGLTs). This study aimed to elucidate the role of these distinct mechanisms in glucose-stimulated GLP-1 secretion. Methods: Glucose uptake into L cells (either GLUTag cells or cells in primary cultures, using a new transgenic mouse model combining proglucagon promoter-driven Cre recombinase with a ROSA26tdRFP reporter) was monitored with the FLIIPglu-700μδ6 glucose sensor. Effects of pharmacological and genetic interference with SGLT1 or facilitative glucose transport (GLUT) on intracellular glucose accumulation and metabolism (measured by NAD(P)H autofluorescence), cytosolic Ca (monitored with Fura2) and GLP-1 secretion (assayed by ELISA) were assessed. Results: L cell glucose uptake was dominated by GLUT-mediated transport, being abolished by phloretin but not phloridzin. NAD(P)H autofluorescence was glucose dependent and enhanced by a glucokinase activator. In GLUTag cells, but not primary L cells, phloretin partially impaired glucose-dependent secretion, and suppressed an amplifying effect of glucose under depolarising high K conditions. The key importance of SGLT1 in GLUTag and primary cells was evident from the impairment of secretion by phloridzin or Sglt1 knockdown and failure of glucose to trigger cytosolic Ca elevation in primary L cells from Sglt1 knockout mice. Conclusions/interpretation: SGLT1 acts as the luminal glucose sensor in L cells, but intracellular glucose concentrations are largely determined by GLUT activity. Although L cell glucose metabolism depends partially on glucokinase activity, this plays only a minor role in glucose-stimulated GLP-1 secretion. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the gastroprotective activity of carvacrol, a monoterpene present in essential oils from several species of medicinal and aromatic plants, by using different models of acute gastric lesions in rodents and also evaluate possible mechanisms involved in this action. For this study, absolute ethanol-, acidified ethanol-, ischemia and reperfusion-, and nonsteroidal anti-inflammatory drug-induced models of gastric lesions in mice and rats were used. The roles of nonprotein sulfhydryl groups, catalase, nitric oxide (NO), ATP-sensitive potassium channels (K channels), and prostaglandins in carvacrol-induced gastroprotective effect were investigated. In addition, the effects of carvacrol on gastric secretion and mucus in pylorus-ligated rats were also determined. The results of the present study demonstrated that carvacrol promoted a marked gastroprotection in all models investigated, possibly mediated by endogenous prostaglandins, increase of mucus production, K channels opening, NO synthase activation, and antioxidant properties. These findings markedly substantiate further studies to investigate the therapeutic potential of carvacrol as an effective gastroprotective agent and its safety profile in medicinal use. [ABSTRACT FROM AUTHOR]

Clarification of the molecular mechanisms of insulin secretion is crucial for understanding the pathogenesis and pathophysiology of diabetes and for development of novel therapeutic strategies for the disease. Insulin secretion is regulated by various intracellular signals generated by nutrients and hormonal and neural inputs. In addition, a variety of glucose-lowering drugs including sulfonylureas, glinide-derivatives, and incretin-related drugs such as dipeptidyl peptidase IV (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists are used for glycaemic control by targeting beta cell signalling for improved insulin secretion. There has been a remarkable increase in our understanding of the basis of beta cell signalling over the past two decades following the application of molecular biology, gene technology, electrophysiology and bioimaging to beta cell research. This review discusses cell signalling in insulin secretion, focusing on the molecular targets of ATP, cAMP and sulfonylurea, an essential metabolic signal in glucose-induced insulin secretion (GIIS), a critical signal in the potentiation of GIIS, and the commonly used glucose-lowering drug, respectively. [ABSTRACT FROM AUTHOR]

Purpose: Despite favorable metabolic and vascular effects, thiazolidinedione (TZD) drugs have not convincingly reduced cardiovascular mortality in clinical trials, raising the possibility of countervailing, off-target effects. We previously showed that TZDs block cardiac ATP-sensitive potassium (K) channels in pigs. In this study, we investigated whether TZDs affect onset, spectral characteristics, and mortality of ischemic ventricular fibrillation (VF) and whether such effects are recapitulated by a non-selective K blocker (glyburide) or a mitochondrial K blocker (5-hydroxydecanoate). Methods: A total of 121 anesthetized pigs were pre-treated with TZD (pioglitazone or rosiglitazone, 1 mg/kg IV, resulting in clinically relevant plasma concentrations), glyburide (1 mg/kg IV), 5-hydroxydecanoate (5 mg/kg IV) or inert vehicle. Ischemia was produced by occlusion of the left anterior descending coronary artery. In a subset of pigs treated with rosiglitazone or vehicle, ischemic preconditioning was performed. Results: VF developed in all but 6 pigs. In non-preconditioned pigs, onset of VF occurred sooner with pioglitazone (11 ± 3 min, p < 0.05) or rosiglitazone (14 ± 3 min, p = 0.06) than with vehicle (20 ± 2 min). Defibrillation of VF was successful in 44 % of pigs treated with vehicle, compared with 0 % with pioglitazone ( p = 0.057) and 33 % with rosiglitazone (NS). After ischemic preconditioning, defibrillation was successful in 62 % of pigs treated with vehicle, compared with 26% treated with rosiglitazone ( p = 0.03). TZDs attenuated slowing of conduction due to ischemia and shifted ECG power spectra during VF toward higher frequencies. All effects of TZDs were recapitulated by glyburide, but not by 5-hydroxydecanoate, supporting an interaction of TZDs with the sarcolemmal K channel. Conclusion: In a porcine model, TZDs promote onset and increase mortality of ischemic VF, associated with alterations of conduction and VF spectral characteristics. Similar effects in a clinical setting might adversely impact cardiovascular mortality. [ABSTRACT FROM AUTHOR]

Intracellular Ca signals underlying conducted vasoconstriction to local application of a brief depolarizing KCl stimulus was investigated in rat mesenteric terminal arterioles (<40 μm). Using a computer model of an arteriole segment comprised of coupled endothelial cells (EC) and vascular smooth muscle cells (VSMC) simulations of both membrane potential and intracellular [Ca] were performed. The 'characteristic' length constant, λ, was approximated using a modified cable equation in both experiments and simulations. We hypothesized that K conductance in the arteriolar wall limit the electrotonic spread of a local depolarization along arterioles by current dissipation across the VSMC plasma membrane. Thus, we anticipated an increased λ by inhibition of voltage-activated K channels. Application of the BK channel blocker iberiotoxin (100 nM) onto mesenteric arterioles in vitro and inhibition of BK channel current in silico increased λ by 34% and 32%, respectively. Similarly, inhibition of K channels in vitro (4-aminopyridine, 1 mM) or in silico increased λ by 41% and 21%, respectively. Immunofluorescence microscopy demonstrated expression of BK, Kv1.5, Kv2.1, but not Kv1.2, in VSMCs of rat mesenteric terminal arterioles. Our results demonstrate that inhibition of voltage-activated K channels enhance vascular-conducted responses to local depolarization in terminal arterioles by increasing the membrane resistance of VSMCs. These data contribute to our understanding of how differential expression patterns of voltage-activated K channels may influence conducted vasoconstriction in small arteriolar networks. This finding is potentially relevant to understanding the compromised microcirculatory blood flow in systemic vascular diseases such as diabetes mellitus and hypertension. [ABSTRACT FROM AUTHOR]

The membrane potential ( V) of beta-cells oscillates at glucose concentrations between ~6 and 25 mM, i.e. burst phases with action potentials alternate with silent interburst phases generating so-called slow waves. The slow waves drive oscillations of the cytosolic Ca concentration ([Ca]) and insulin secretion. The length of the bursts correlates with the amount of insulin release. Thus, the fraction of plateau phase (FOPP), i.e. the percentage of time with burst activity, is an excellent marker for beta-cell function and metabolic integrity. Extracellular voltage changes of mouse islets were measured using a microelectrode array (MEA) allowing the detection of burst and interburst phases. At a non-stimulating glucose concentration (3 mM) no electrical activity was detectable while bursting was continuous at 30 mM. The glucose concentration-response (determined as FOPP) curve revealed half-maximal stimulation at 12 ± 1 mM (Hill equation fit). The signal was sensitive to K channel modulators, e.g. tolbutamide or diazoxide. Simultaneous recordings of electrical activity and [Ca] revealed congruent bursts and peaks, respectively. The extracellular recordings are in perfect agreement with more time-consuming intracellular electrical recordings. The results provide a 'proof-of-principle' for detection of beta-cell slow waves and determination of the FOPP using extracellular electrodes in a MEA-based system. The method is facile and provides the capability to study the effects of modulators of beta-cell function including possible anti-diabetic drugs in real time. Moreover, the method may be useful for checking the metabolic integrity of human donor islets prior to transplantation. [ABSTRACT FROM AUTHOR]

Sulfonylurea receptors (SURs) form an integral part of the ATP-sensitive potassium (K) channel complex that is present in most excitable cell types. K channels couple cellular metabolism to electrical activity and provide a wide range of cellular functions including stimulus secretion coupling in pancreatic β cells. K channels are composed of SURs and inward rectifier potassium channel (Kir6.x) subunits encoded by the ABCC8/9 and KCNJ8/11 genes, respectively. Recent advances in the genetics, molecular biology, and pharmacology of SURs have led to an increased understanding of these channels in the etiology and treatment of rare genetic insulin secretory disorders. Furthermore, common genetic variants in these genes are associated with an increased risk for type 2 diabetes. In this review we summarize the molecular biology, pharmacology, and physiology of SURs and K channels, highlighting recent advances in their genetics and understanding of rare insulin secretory disorders and susceptibility to type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Nifekalant and azimilide, Class III antiarrhythmic agents, block the human ether-à-go-go-related gene K+ (hERG) channel. However, when a depolarizing membrane potential is applied, they also increase the current at low potentials by shifting its activation curve towards hyperpolarizing voltages. This phenomenon is called ‘facilitation’. In this study, we tried to address the mechanism underlying the facilitation by analyzing the effects of various compounds on hERG expressed in Xenopus oocytes. Like nifekalant, amiodarone, quinidine and carvedilol, but not by dofetilide, caused the current facilitation of hERG, suggesting that the facilitation is a common effect to a subset of hERG blockers. As the concentration of each compound was increased, the total hERG current was suppressed progressively, while the current at low potentials was augmented. Activation curves of the remaining hERG current in the facilitation condition could be described as the sum of two Boltzmann functions reflecting two populations of hERG currents having different activation curves. The voltage shift in the activation curve from control was constant for each compound even at different concentrations; −31mV in amiodarone, −27mV in nifekalant, −17mV in quinidine and −12mV in carvedilol. Therefore, the facilitation is based on the appearance of hERG whose voltage-dependence for the activation is shifted towards hyperpolarizing voltages. [Copyright &y& Elsevier]

Lead (Pb) is a trace metal element in the human body. In order to understand the hazard mechanism of the elevated blood lead level on the human body, the effect of Pb on the human ether-à-go-go-related gene (hERG) K channel in the HEK 293 cell was investigated for the first time using whole-cell patch clamp technique, molecular dynamics simulation, and quantum chemistry calculation methods. We found that Pb obviously inhibits the current of the hERG K channel, and delays the 'activation' and 'deactivation' of the hERG K channel, indicating that Pb evidently decreases the function of the K channel in the cell. The effect is increased with increasing the concentration of Pb. When the concentration of Pb is 400 μg L, the function of the K channel is entirely lost. The results from the molecular dynamics simulation and quantum chemistry calculation indicated that Pb can coordinate with the oxygen/sulfur atoms in the K channel protein, leading to the decrease in the function of the K channel. According to the experimental results, we suggested that once the K channel in the human body was irreversibly inactivated by Pb, it would affect the treatment and prognosis of Pb intoxication. [ABSTRACT FROM AUTHOR]

In hypertonic solutions made by adding nonelectrolytes, K(+) channels of squid giant axons opened at usual asymmetrical K(+) concentrations in two different time courses; an initial instantaneous activation (I (IN)) and a sigmoidal activation typical of a delayed rectifier K(+) channel (I (D)). The current-voltage relation curve for I (IN) was fitted well with Goldman equation described with a periaxonal K(+) concentration at the membrane potential above -10 mV. Using the activation-voltage curve obtained from tail currents, K(+) channels for I (IN) are confirmed to activate at the membrane potential that is lower by 50 mV than those for I (D). Both I (IN) and I (D) closed similarly at the holding potential below -100 mV. The logarithm of I (IN)/I (D) was linearly related with the osmolarity for various nonelectrolytes. Solute inaccessible volumes obtained from the slope increased with the nonelectrolyte size from 15 to 85 water molecules. K(+) channels representing I (D) were blocked by open channel blocker tetra-butyl ammonium (TBA) more efficiently than in the absence of I (IN), which was explained by the mechanism that K(+) channels for I (D) were first converted to those for I (IN) by the osmotic pressure and then blocked. So K(+) channels for I (IN) were suggested to be derived from the delayed rectifier K(+) channels. Therefore, the osmotic pressure is suggested to exert delayed-rectifier K(+) channels to open in shrinking rather hydrophilic flexible parts outside the pore than the pore itself, which is compatible with the recent structure of open K(+) channel pore. [ABSTRACT FROM AUTHOR]

In an attempt to explore unknown K(+) channels in mammalian cells, especially ATP-sensitive K(+) (K(ATP)) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian K(ATP) channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K(+) channel current that was inwardly rectified and had a reversal potential close to K(+) equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific K(ATP) channel openers or blockers did not alter the K(+) current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of K(ATP) channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K(+) currents were inhibited by protopine (a nonspecific K(+) channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K(+) channel in mammalian cells. [ABSTRACT FROM AUTHOR]

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

In this study, we cloned a cDNA for the K channel β subunit from the halophyte Puccinellia tenuiflora and named it KPutB1. KPutB1 was preferentially expressed in the roots and was transiently induced by K-starvation, salt stress, or the combination of both stresses. By yeast two-hybrid assay, we demonstrated that KPutB1 interacts with PutAKT1, α subunit of an AKT1-type K channel of P. tenuiflora. The functional relevance of this interaction on K-nutrition was investigated by co-expression experiments in yeast under various ionic conditions, and K channel α and β subunit homologues from rice ( OsAKT1 and KOB1, respectively) were included for comparison. Yeast co-expressing PutAKT1 and the β subunits ( KPutB1 and KOB1) had better growth and higher K-uptake ability than yeast expressing PutAKT1 alone. In contrast, yeast co-expressing the β subunits ( KPutB1 and KOB1) with OsAKT1 had slower growth and lower K uptake than yeast expressing OsAKT1 alone. Arabidopsis plants over-expressing the K channel β subunit of P. tenuiflora or rice showed increased shoot K content and decreased root Na content under control, 75 mM NaCl, and K-starvation stress conditions. These results suggest that ectopic expression of the K channel β subunit could alter K and Na homeostasis in plants. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury. [Copyright &y& Elsevier]