Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of β-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.     

Role of mitochondrial ATP-sensitive potassium channels on fatigue in mouse muscle fibers

The role of mitochondrial K_ATP (mitoK_ATP) channels on muscle fatigue was assessed in adult mouse skeletal muscle bundles. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22 °C) using a force transducer and the fluorescent indicator CM-H₂DCFDA. We found that opening mitoK_ATP channels with diazoxide (100 μM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoK_ATP channel blocker 5-hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor N^G-nitro-l-arginine methyl ester hydrochloride (l-NAME) but was not accompanied by a change in the rate of ROS production during fatigue. A physiological role of mitoK_ATP channels on muscle fatigue is proposed.     

Mitochondrial ATP-sensitive potassium channels in surgical cardioprotection

ATP-sensitive potassium channels allow for the coupling of membrane potential to cellular metabolic status. Two K(ATP) channel subtypes coexist in the myocardium with one subtype located in the sarcolemma membrane and the other in the inner membrane of the mitochondria. The ATP-sensitive potassium channels can be pharmacologically modulated by a family of structurally diverse agents of varied potency and selectivity, collectively known as potassium channel openers and blockers. Sufficient evidence exists to indicate that the ATP-sensitive potassium channels and in particular the mitochondrial ATP-sensitive potassium channels play an important role both as a trigger and an effector in surgical cardioprotection. In this review, the biochemistry and specificity of the ATP-sensitive potassium channels is examined in relation to surgical cardioprotection.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.     

Assembly limits the pharmacological complexity of ATP-sensitive potassium channels

ATP-sensitive potassium channels (K(ATP) channels) are formed from an octameric complex of an inwardly rectifying K(+) channel (Kir6.1, Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, and SUR2B). In this study we have attempted to address the question of whether SUR heteromultimers can form using a combination of biochemical and electrophysiological approaches. We have constructed monoclonal stable lines in HEK293 cells co-expressing Kir6.2 with SUR1 and SUR2A. Using coimmunoprecipitation analysis with SUR isotype-specific antibodies two biochemical populations are distinguished, one containing SUR1 and the other SUR2A. It is not possible to detect immune complexes containing both SUR1 and SUR2A. Functional studies were undertaken and whole cell membrane currents were studied using the patch clamp. Concentrations of sulfonylureas and potassium channel openers were determined that selectively inhibited or activated SUR1/Kir6.2 and SUR2A/Kir6.2. In the cell line expressing SUR1/SUR2AKir6.2 we were unable to demonstrate a population of channels with unique pharmacological properties. Thus we conclude from these studies that heteromultimeric channel complexes containing both SUR1 and SUR2A are not formed, suggesting an incompatibility between different SUR subtypes. This incompatibility limits the pharmacological complexity of K(ATP) channels that may be observed in native tissues.     

Dexmedetomidine directly inhibits vascular ATP-sensitive potassium channels

AimsDexmedetomidine is reported to have an effect on peripheral vasoconstriction; however, the exact mechanisms underlying this process are unclear. In this study, we hypothesized that dexmedetomidine-induced inhibition of vascular ATP-sensitive K⁺ (K_ATP) channels may be associated with this vasoconstriction. To test this hypothesis, we investigated the effects of dexmedetomidine on vascular K_ATP-channel activity at the single-channel level.Main methodsWe used cell-attached and inside-out patch-clamp configurations to examine the effects of dexmedetomidine on the activities of native rat vascular K_ATP channels, recombinant K_ATP channels with different combinations of various inwardly rectifying potassium channels (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor subunits (SUR1, 2A, 2B), and SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit, namely, Kir6.2ΔC36 channels.Key findingsDexmedetomidine was observed to inhibit the native rat vascular K_ATP channels in both cell-attached and inside-out configurations. This drug also inhibited the activity of all types of recombinant SUR/Kir6.0 K_ATP channels as well as Kir6.2ΔC36 channels with equivalent potency.SignificanceThese results indicate that dexmedetomidine directly inhibits K_ATP channels through the Kir6.0 subunit.     

Expression of ATP-sensitive potassium channels in human pregnant myometrium

Background  

Potassium channels play critical roles in the regulation of cell membrane potential, which is central to the excitability of myometrium. The ATP-sensitive potassium (KATP) channel is one of the most abundant potassium channels in myometrium. The objectives of this study were to investigate the protein expression of KATP channel in human myometrium and determine the levels of KATP channel in lower and upper segmental myometrium before and after onset of labour.     

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.     

ATP敏感的钾通道与预适应心肌保护作用

缺血、药物等多种因素产生的预适应现象都具有显著的心肌保护作用。ATP敏感的钾通道是介导预适应保护作用的重要环节。目前多数研究结果表明是线粒体而非质膜ATP敏感的钾通道介导了预适应的保护作用,但它是否为此过程的最终效应器尚有待更多更深入的研究,未来线粒体ATP敏感钾通道的克隆和调控机制的揭示,将是从根本上解决这一问题的关键。     

Inhibitors of ATP-sensitive potassium channels stimulate intestinal cholecystokinin secretion

Recently, a role for adenosine 5′-triphosphate(ATP)-sensitive potassium channels in the regulation of cholecystokinin (CCK) secretion has been described in STC-1 cells, an intestinal CCK-secreting cell line. To examine whether a similiar mechanism might participate in the regulation of hormone secretion from native CCK cells, the effects of two established inhibitors of ATP-sensitive potassium channels (e.g. glucose, disopyramide) were examined on CCK release from dispersed murine intestinal cells. Both glucose and disopyramide were found to stimulate CCK secretion. Furthermore, CCK release induced by glucose was inhibited by the calcium channel blocker diltiazem. It is concluded that, ATP-sensitive potassium channels may play a role in the regulation of intestinal CCK secretion.     

Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel was discovered more than a decade ago. Since then, several pharmacological studies have identified agents that target this channel some of which selectively target mitoK(ATP). These and other studies have also suggested that mitoK(ATP) plays a key role in the process of ischemic preconditioning (IPC) and prevention of apoptosis. The mechanism by which mitoK(ATP) exerts its protective effects is unclear, however, changes in mitochondrial Ca(2+) uptake and levels of reactive oxygen species, and mitochondrial matrix swelling are believed to be involved. Despite major advances, several important issues regarding mitoK(ATP) remain unanswered. These questions include, but are not limited to: the molecular structure of mitoK(ATP), the downstream and upstream mechanisms that leads to IPC and cell death, and the pharmacological profile of the channel. This review attempts to provide an up-to-date overview of the role of mitoK(ATP) in cardioprotection.     

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

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

ATP-sensitive potassium channels: metabolic sensing and cardioprotection.

The cardiovascular system operates under a wide scale of demands, ranging from conditions of rest to extreme stress. How the heart muscle matches rates of ATP production with utilization is an area of active investigation. ATP-sensitive potassium (K(ATP)) channels serve a critical role in the orchestration of myocardial energetic well-being. K(ATP) channel heteromultimers consist of inwardly-rectifying K(+) channel 6.2 and ATP-binding cassette sulfonylurea receptor 2A that translates local ATP/ADP levels, set by ATPases and phosphotransfer reactions, to the channel pore function. In cells in which the mobility of metabolites between intracellular microdomains is limited, coupling of phosphotransfer pathways with K(ATP) channels permits a high-fidelity transduction of nucleotide fluxes into changes in membrane excitability, matching energy demands with metabolic resources. This K(ATP) channel-dependent optimization of cardiac action potential duration preserves cellular energy balance at varying workloads. Mutations of K(ATP) channels result in disruption of the nucleotide signaling network and generate a stress-vulnerable phenotype with excessive susceptibility to injury, development of cardiomyopathy, and arrhythmia. Solving the mechanisms underlying the integration of K(ATP) channels into the cellular energy network will advance the understanding of endogenous cardioprotection and the development of strategies for the management of cardiovascular injury and disease progression.     

Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a pivotal role in early and late ischemic preconditioning, but the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in rat hearts using confocal microscopy, immunofluorescence, and Western blot analysis. The green fluorescent probe glibenclamide-BODIPY was colocalized with the red fluorescent mitochondrial marker MitroTracker Red in isolated ventricular myocytes and in ventricular myocyte mitochondria, indicating the presence of sulfonylurea receptors (SURs) in the mitochondria. Anti-Kir6.1, anti-Kir6.2, and anti-SUR2 immunofluorescence was colocalized with that of MitoTracker Red in isolated mitochondria, suggesting that Kir6.1, Kir6.2, and SUR2 subunits are present in the mitochondria. Similarly, Kir6.1 (approximately 46 kDa), Kir6.2 (approximately 46 and approximately 40 kDa), and SUR2 (approximately 140 kDa) proteins were found to be expressed in mitochondria using Western blot analysis. By contrast, SUR1 was not present in mitochondria. These results suggest that mitoKATP channels in rat hearts might comprise a combination of Kir6.1, Kir6.2, and SUR2 subunits.     

Localization and function of ATP-sensitive potassium channels in human skeletal muscle

The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique or the sucrose-gradient technique in combination with Western blotting demonstrated that the KATP channels are mainly located in the sarcolemma. This localization was confirmed by immunohistochemical measurements. With the microdialysis technique, it was demonstrated that local application of the KATP channel inhibitor glibenclamide reduced (P < 0.05) interstitial K+ at rest from approximately 4.5 to 4.0 mM, whereas the concentration in the control leg remained constant. Glibenclamide had no effect on the interstitial K+ accumulation during knee-extensor exercise at a power output of 60 W. In contrast to in vitro conditions, the present study demonstrated that under in vivo conditions the KATP channels are active at rest and contribute to the accumulation of interstitial K+.     

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle

In mildly hyperosmotic medium, activation of the Na⁺-K⁺-2Cl^- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (K_ATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a K_ATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of K_ATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of K_ATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions. protein kinase A; glibenclamide; glycolysis; Na⁺-K⁺-2Cl^- cotransporter; Kir6.2