SNAP-251–180 enhances insulin secretion by blocking Kv2.1 channels in rat pancreatic islet β-cells

Voltage-gated outward K⁺ currents from pancreatic islet β-cells are known to repolarize the action potential during a glucose stimulus, and consequently to modulate Ca²⁺ entry and insulin secretion. The voltage gated K⁺ (Kv) channel, Kv2.1, which is expressed in rat islet β-cells, mediates over 60% of the Kv outward K⁺ currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-25_1–180 (S180), an N-terminal SNAP-25 domain, but not SNAP-25_1–206 (S206), inhibits Kv current and enhances glucose-dependent insulin secretion from rat pancreatic islet β-cells, and furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner.     

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

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 Ca²⁺/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 (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP 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 K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

A Role for Cytosolic Isocitrate Dehydrogenase as a Negative Regulator of Glucose Signaling for Insulin Secretion in Pancreatic ?-Cells

Cytosolic NADPH may act as one of the signals that couple glucose metabolism to insulin secretion in the pancreatic ß-cell. NADPH levels in the cytoplasm are largely controlled by the cytosolic isoforms of malic enzyme and isocitrate dehydrogenase (IDHc). Some studies have provided evidence for a role of malic enzyme in glucose-induced insulin secretion (GIIS) via pyruvate cycling, but the role of IDHc in ß-cell signaling is unsettled. IDHc is an established component of the isocitrate/α–ketoglutarate shuttle that transfers reducing equivalents (NADPH) from the mitochondrion to the cytosol. This shuttle is energy consuming since it is coupled to nicotinamide nucleotide transhydrogenase that uses the mitochondrial proton gradient to produce mitochondrial NADPH and NAD⁺ from NADP⁺ and NADH. To determine whether flux through IDHc is positively or negatively linked to GIIS, we performed RNAi knockdown experiments in ß-cells. Reduced IDHc expression in INS 832/13 cells and isolated rat islet ß-cells resulted in enhanced GIIS. This effect was mediated at least in part via the K_ATP-independent amplification arm of GIIS. IDHc knockdown in INS 832/13 cells did not alter glucose oxidation but it reduced fatty acid oxidation and increased lipogenesis from glucose. Metabolome profiling in INS 832/13 cells showed that IDHc knockdown increased isocitrate and NADP⁺ levels. It also increased the cellular contents of several metabolites linked to GIIS, in particular some Krebs cycle intermediates, acetyl-CoA, glutamate, cAMP and ATP. The results identify IDHc as a component of the emerging pathways that negatively regulate GIIS.     

The Inhibitory Effects of Ca2+ Channel Blocker Nifedipine on Rat Kv2.1 Potassium Channels

It is well documented that nifedipine, a commonly used dihydropyridine Ca²⁺ channel blocker, has also significant interactions with voltage-gated K⁺ (Kv) channels. But to date, little is known whether nifedipine exerted an action on Kv2.1 channels, a member of the Shab subfamily with slow inactivation. In the present study, we explored the effects of nifedipine on rat Kv2.1 channels expressed in HEK293 cells. Data from whole-cell recording showed that nifedipine substantially reduced Kv2.1 currents with the IC₅₀ value of 37.5 ± 5.7 μM and delayed the time course of activation without effects on the activation curve. Moreover, this drug also significantly shortened the duration of inactivation and deactivation of Kv2.1 currents in a voltage-dependent manner. Interestingly, the half-maximum inactivation potential (V _1/2) of Kv2.1 currents was -11.4 ± 0.9 mV in control and became -38.5 ± 0.4 mV after application of 50 μM nifedipine. The large hyperpolarizing shift (27 mV) of the inactivation curve has not been reported previously and may result in more inactivation for outward delayed rectifier K⁺ currents mediated by Kv2.1 channels at repolarization phases. The Y380R mutant significantly increased the binding affinity of nifedipine to Kv2.1 channels, suggesting an interaction of nifedipine with the outer mouth region of this channel. The data present here will be helpful to understand the diverse effects exerted by nifedipine on various Kv channels.     

Mitochondrial Metabolism of Pyruvate Is Essential for Regulating Glucose-stimulated Insulin Secretion

It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (K_ATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP⁺ ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.     

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet β-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic α-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic β-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP⁺ ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.     

Nesfatin-1 inhibits voltage gated K+ channels in pancreatic beta cells

The anorexigenic neuropeptide NEFA/nucleobindin 2 (NUCB2)/nesfatin-1-containing neurons are distributed in the brain regions involved in feeding regulation. In spite of the growing knowledge of its physiological functions through extensive studies, its molecular mechanism of reaction, including its receptor, remains unknown. NUCB2/nesfatin-1 is also involved in various peripheral regulations, including glucose homeostasis. In pancreatic beta-cells, NUCB2/nesfatin-1 is reported to enhance glucose-stimulated insulin secretion (GSIS) but its exact mechanism remains unknown.To clarify this mechanism, we measured the effect of nesfatin-1 on the electrical activity of pancreatic beta-cells. Using mouse primary beta cells, we measured changes in the ATP-sensitive K⁺ (K_ATP) channel current, the voltage-gated K⁺ (Kv) channel current, and insulin secretion upon application of nesfatin-1.Nesfatin-1 inhibited the Kv channel, but K_ATP channel activity was unaffected. Nesfatin-1 enhanced insulin secretion to a same level as Kv channel blocker tetraethylammonium (TEA). The effect was not further enhanced when nesfatin-1 and TEA were applied simultaneously. The inhibition binding assay with [¹²⁵I]nesfatin-1 in Kv2.1 channels, major contributor of Kv current in beta cell, expressing HEK239 cells indicated the binding of nesfatin-1 on Kv2.1 channel.Because Kv channel inhibition enhances insulin secretion under high glucose conditions, our present data suggest a possible mechanism of nesfatin-1 on enhancing GSIS through regulation of ion channels rather than its unidentified receptor.     

A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.     

Inhibition of Pancreatic β-Cell Ca2+/Calmodulin-dependent Protein Kinase II Reduces Glucose-stimulated Calcium Influx and Insulin Secretion,Impairing Glucose Tolerance

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is caused by Ca²⁺ entry via voltage-dependent Ca²⁺ channels. CaMKII is a key mediator and feedback regulator of Ca²⁺ signaling in many tissues, but its role in β-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by β-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of K_ATP and voltage-gated potassium channels. However, glucose-stimulated Ca²⁺ entry via voltage-dependent Ca²⁺ channels is reduced in islet β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-cell cytoplasmic Ca²⁺ and of endoplasmic reticulum Ca²⁺ stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca²⁺ sensor with a key role as a feed-forward stimulator of β-cell Ca²⁺ signals that enhance GSIS under physiological and pathological conditions.     

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable,Depending on the Relative Expression Level

Kv4 is a voltage-gated K⁺ channel, which underlies somatodendritic subthreshold A-type current (I_SA) and cardiac transient outward K⁺ (I_to) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K⁺ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.     

Forskolin Suppresses Delayed-Rectifier K+ Currents and Enhances Spike Frequency-Dependent Adaptation of Sympathetic Neurons

In signal transduction research natural or synthetic molecules are commonly used to target a great variety of signaling proteins. For instance, forskolin, a diterpene activator of adenylate cyclase, has been widely used in cellular preparations to increase the intracellular cAMP level. However, it has been shown that forskolin directly inhibits some cloned K⁺ channels, which in excitable cells set up the resting membrane potential, the shape of action potential and regulate repetitive firing. Despite the growing evidence indicating that K⁺ channels are blocked by forskolin, there are no studies yet assessing the impact of this mechanism of action on neuron excitability and firing patterns. In sympathetic neurons, we find that forskolin and its derivative 1,9-Dideoxyforskolin, reversibly suppress the delayed rectifier K⁺ current (I_KV). Besides, forskolin reduced the spike afterhyperpolarization and enhanced the spike frequency-dependent adaptation. Given that I_KV is mostly generated by Kv2.1 channels, HEK-293 cells were transfected with cDNA encoding for the Kv2.1 α subunit, to characterize the mechanism of forskolin action. Both drugs reversible suppressed the Kv2.1-mediated K⁺ currents. Forskolin inhibited Kv2.1 currents and I_KV with an IC₅₀ of ~32 μM and ~24 µM, respectively. Besides, the drug induced an apparent current inactivation and slowed-down current deactivation. We suggest that forskolin reduces the excitability of sympathetic neurons by enhancing the spike frequency-dependent adaptation, partially through a direct block of their native Kv2.1 channels.     

Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.     

Evidence for presence and functional effects of Kv1.1 channels in β-cells: general survey and results from mceph/mceph mice

Background

Voltage-dependent K⁺ channels (Kv) mediate repolarisation of β-cell action potentials, and thereby abrogate insulin secretion. The role of the Kv1.1 K⁺ channel in this process is however unclear. We tested for presence of Kv1.1 in different species and tested for a functional role of Kv1.1 by assessing pancreatic islet function in BALB/cByJ (wild-type) and megencephaly (mceph/mceph) mice, the latter having a deletion in the Kv1.1 gene.

Methodology/Principal Findings

Kv1.1 expression was detected in islets from wild-type mice, SD rats and humans, and expression of truncated Kv1.1 was detected in mceph/mceph islets. Full-length Kv1.1 protein was present in islets from wild-type mice, but, as expected, not in those from mceph/mceph mice. Kv1.1 expression was localized to the β-cell population and also to α- and δ-cells, with evidence of over-expression of truncated Kv1.1 in mceph/mceph islets. Blood glucose, insulin content, and islet morphology were normal in mceph/mceph mice, but glucose-induced insulin release from batch-incubated islets was (moderately) higher than that from wild-type islets. Reciprocal blocking of Kv1.1 by dendrotoxin-K increased insulin secretion from wild-type but not mceph/mceph islets. Glucose-induced action potential duration, as well as firing frequency, was increased in mceph/mceph mouse β-cells. This duration effect on action potential in β-cells from mceph/mceph mice was mimicked by dendrotoxin-K in β-cells from wild-type mice. Observations concerning the effects of both the mceph mutation, and of dendrotoxin-K, on glucose-induced insulin release were confirmed in pancreatic islets from Kv1.1 null mice.

Conclusion/Significance

Kv1.1 channels are expressed in the β-cells of several species, and these channels can influence glucose-stimulated insulin release.     

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K⁺ channels was investigated, in connection with the known functional diversity of K⁺ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K⁺ currents could be approximated by the algebraic sum of the 2 K⁺ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K⁺ channels.     

Critical role of gap junction coupled KATP channel activity for regulated insulin secretion

Pancreatic β-cells secrete insulin in response to closure of ATP-sensitive K⁺ (K_ATP) channels, which causes membrane depolarization and a concomitant rise in intracellular Ca²⁺ (Ca_i). In intact islets, β-cells are coupled by gap junctions, which are proposed to synchronize electrical activity and Ca_i oscillations after exposure to stimulatory glucose (>7 mM). To determine the significance of this coupling in regulating insulin secretion, we examined islets and β-cells from transgenic mice that express zero functional K_ATP channels in approximately 70% of their β-cells, but normal K_ATP channel density in the remainder. We found that K_ATP channel activity from approximately 30% of the β-cells is sufficient to maintain strong glucose dependence of metabolism, Ca_i, membrane potential, and insulin secretion from intact islets, but that glucose dependence is lost in isolated transgenic cells. Further, inhibition of gap junctions caused loss of glucose sensitivity specifically in transgenic islets. These data demonstrate a critical role of gap junctional coupling of K_ATP channel activity in control of membrane potential across the islet. Control via coupling lessens the effects of cell–cell variation and provides resistance to defects in excitability that would otherwise lead to a profound diabetic state, such as occurs in persistent neonatal diabetes mellitus.     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.     

Geniposide Regulates Glucose-Stimulated Insulin Secretion Possibly through Controlling Glucose Metabolism in INS-1 Cells

Glucose-stimulated insulin secretion (GSIS) is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM). Although the K_ATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.     

Overexpression of the Large-Conductance,Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes

Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca²⁺-activated K⁺ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca²⁺-sensitive K⁺ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K⁺ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.