Dual regulation of the ATP-sensitive potassium channel by activation of cGMP-dependent protein kinase

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.     

Functional modulation of the ATP-sensitive potassium channel by extracellular signal-regulated kinase-mediated phosphorylation

ATP-sensitive potassium (K(ATP)) channels play an important role in controlling insulin secretion and vascular tone as well as protecting neurons under metabolic stress. We have previously demonstrated that stimulation of the K(ATP) channel by nitric oxide (NO) requires activation of Ras- and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. However, the mechanistic link between ERK and the K(atp) channel remained unknown. To investigate how ERK modulates the function of K(ATP) channels, we performed single-channel recordings in combination with site-directed mutagenesis. The Kir6.2/SUR1 channel, a neuronal K(ATP) channel isoform, was expressed in human embryonic kidney (HEK) 293 cells by transient transfection. Direct application of the activated ERK2 to the cytoplasmic surface of excised, inside-out patches markedly enhanced the single-channel activity of Kir6.2/SUR1 channels. The normalized open probability (NPo) and opening frequency were significantly increased, whereas the mean closed duration was reduced. The single-channel conductance level was not affected. The ERK2-induced stimulation of Kir6.2/SUR1 channels was prevented by heat-inactivation of the enzyme. Furthermore, alanine substitutions of T341 and S385 to disrupt the potential ERK phosphorylation sites present in the Kir6.2 subunit significantly abrogated the stimulatory effects of ERK2, while aspartate substitutions of T341 and S385 to mimic the (negative) charge effect of phosphorylation rendered a small yet significant reduction in the ATP sensitivity of the channel. Taken together, here we report for the first time that ERK2/MAPK activates neuronal-type K(ATP) channels, and this stimulation requires ERK phosphorylation of the Kir6.2 subunit at T341 and S385 residues. The ERK2-induced K(ATP) channel stimulation can be accounted for by changes in channel gating that destabilize the closed states and by reduction in the ATP sensitivity. As Kir6.2 is the pore-forming subunit of K(ATP) channels, ERK2-mediated phosphorylation may represent a common mechanism for K(ATP) channel regulation in different tissues.     

Modulation of muscle contractility during fatigue and recovery by ATP-sensitive potassium channel

The activity of ATP-sensitive potassium channels of skeletal muscle is controlled by changes in the bioenergetic state of the cell. These channels are inactive in unfatigued muscle and become activated during fatigue. It has been postulated that ATP-sensitive potassium channels shorten the action potential duration, increase the potassium efflux and contribute to the decrease in force during fatigue. Although blocking ATP-sensitive potassium channels during fatigue prolongs the action potential duration and decreases the potassium efflux as expected, it does not affect the rate of fatigue development, as observed from the decrease in tetanic force. Even though such results are not consistent with the hypothesis that ATP-sensitive potassium channels contribute to the decrease in force during fatigue, a reduced capacity of skeletal muscles to recover their tetanic force following fatigue is also observed when ATP-sensitive potassium channels are blocked during fatigue, suggesting that these channels have a myoprotective effect. It is thus possible that removing this myoprotection during fatigue results in deleterious effects which counteract the expected slower decrease in force. However, ATP-sensitive potassium channel openers also fail to affect the rate of fatigue development. Therefore, the results obtained so far do not support the hypothesis that ATP-sensitive potassium channels contribute to the decrease in force during fatigue.     

Polymyxin B has multiple blocking actions on the ATP-sensitive potassium channel in insulin-secreting cells

The action of polymyxin B (0.1 M) on ATP-sensitive K⁺ (K⁺ _ATP) channels in RINm5F insulin-secreting cells was investigated by patch-clamp techniques. Using inside-out patches, open-cells and outside-out patches, polymyxin B was found to block K⁺ _ATP channels by, on average, approximately 90–95% of the initial control level of channel activity. The effects were rapid in onset, sustained and readily reversible. Similar effects were found in patches excised from cells pretreated overnight with 1 M of the phorbol ester phorbol myristate acetate (PMA). External block of channels was associated with a marked decrease in single-channel current amplitude, whereas these effects were not seen when polymyxin B was added to the inside face of the membrane. In patches bathed with internally applied ATP (0.5 mM) and ADP (0.5 mM), polymyxin B inhibited channels but its actions were not reversible upon removal of the compound. However, when the same protocol was undertaken upon cells pre-treated with PMA, the effects of polymyxin B were readily reversed. Our data suggests that polymyxin B is a novel modulator of K⁺ _ATP channels, exhibiting multiple blocking actions that may possibly involve a direct effect upon the channel and indirect effects mediated through the inhibition of endogenous protein kinase(s).To be considered as equal first author.     

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle

Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the K_ATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 M) and ADP (100 M) reversibly increased the channel activity. In the presence of internal Mg²⁺ (1 mM), low concentrations of ADP (<300 M) enhanced channel activity and high concentrations of ADP (>300 M) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg²⁺, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [S] (100 M), which behaved as a weak GDP agonist in the presence of Mg²⁺, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K⁺ channel opener pinacidil was found to activate the K_ATP channel but only in the presence of internal GDP, ADP and GDP [S]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the K_ATP channel.     

Regulation of Hfe by stress factors in BV-2 cells

Mutations in the Hfe gene can be associated with the iron overload disorder known as hemochromatosis. A number of recent studies suggest that carrying an Hfe mutation is a risk factor or genetic modifier for Alzheimer's disease (AD). In AD, Hfe protein expression is induced on cells associated with neuritic plaques and on neurons in the periplaque area. In this study, the factors that may be responsible for induction of Hfe in AD brain were determined using BV-2 cells. Hfe expression was induced by serum deprivation, menadione and beta-amyloid. The labile iron pool was consistently decreased when Hfe expression increased. However, the changes in expression of Hfe appeared independent of the expression of transferrin receptor and ferritin. These data provide insight into the induction of Hfe in AD and indicate that Hfe expression may be a protective function to limit cellular iron exposure during cell stress. These results are the first in a series of studies to understand how mutations in Hfe can be a risk factor for AD.     

雌激素通过KATP通道影响大鼠肠系膜动脉的反应性

目的: 观察雌激素对大鼠肠系膜动脉平滑肌ATP敏感性钾离子通道 (K_ATP 通道)mRNA表达的影响,探讨K_ATP通道在雌激素调节大鼠肠系膜血管反应性中的作用。方法: 雌性SD大鼠48只,体重(100±10) g,随机分为假手术组(sham)、卵巢切除组(Ovx)和卵巢切除后补充雌激素组(Ovx +E)。采取实时荧光定量PCR检测大鼠肠系膜动脉中K_ATP通道mRNA的表达;观察各组大鼠肠系膜动脉对去甲肾上腺素(NE)升压效应的反应性。结果: 与sham组相比,Ovx组大鼠肠系膜动脉K_ATP 通道的Kir6.1及SUR2B亚单位mRNA表达减少(P<0.05),而Ovx+E组则表达增加(P<0.05)。与sham组相比,Ovx组大鼠的血管反应性明显增加(P<0.05),Ovx+E组无明显差异。给予K_ATP通道阻滞剂格列本脲后,sham组和Ovx+E组的动脉反应性增加(P<0.05),Ovx组无明显变化(P>0.05),此时3组间比较无明显差异(P>0.05)。结论: 雌激素可能通过上调肠系膜动脉平滑肌细胞K_ATP通道的表达来降低动脉对去甲肾上腺素升压效应的反应性。     

Effect of the KCNQ potassium channel opener retigabine on single KCNQ2/3 channels expressed in CHO cells

KCNQ2/3 potassium channel subunits were co-expressed in Chinese hamster ovary (CHO) cells and currents through single channels recorded using cell-attached patches. Channels had a similar slope conductance in the presence (8.04 ± 0.02 pS) and absence (7.6 ± 0.01 pS) of 10 μ m retigabine. The mean maximal open probability ( P _o) for single KCNQ2/3 channels was 0.13 ± 0.02, with a half-maximal P _o potential ( V _o) of −28.7 ± 1.4 mV for control recordings. Retigabine increased mean maximal P _o to 0.38 ± 0.04 and produced a hyperpolarising shift of V _o to −40.1 ± 3.4 mV. Single KCNQ2/3 channels have multiple voltage-dependent kinetic components in their activity (C_L-O_S-C_M-O_L-C_S; C = closed, O = open, L = long, S = short, M = medium), giving short, medium and long closed times (τ_CS, τ_CM, τ_CL) and short and long open times (τ_OS and τ_OL). In the presence of retigabine at 0 mV the combined duration and contributions of the longest closed time τ_CL decreased tenfold, while the short and long open times increased fourfold and twofold, respectively. Thus, steady-state kinetics were modified to favour the open channel configuration.     

Age-dependent involvement of ATP-sensitive potassium channel Kir6.2 in hypoxic ventilatory depression of mouse

In order to examine whether ATP-sensitive potassium channel Kir6.2 is involved in hypoxic ventilatory responses, especially in hypoxic ventilatory depression (HVD), and whether the involvement shows age-dependence, we measured the hypoxic ventilatory response in the Kir6.2-knockout mouse (Kir6.2-/-) in an unanesthetized unrestrained state by means of pressure plethysmography in the 2nd and 4th postnatal weeks, and compared the response with that of its wild type counterpart, the C57BL6/J mouse. In the 4th postnatal week, but not in the 2nd week, the Kir6.2-/- exhibited a larger and longer initial augmentation and a weaker subsequent depression of respiratory frequency and ventilation in response to hypoxia (FIO(2)=0.12 in N(2)). These findings suggest that Kir6.2 is involved in HVD of the mouse at a certain point during the postnatal development.     

ATP敏感性钾通道P266T突变对离子通道特性的影响

目的： 通过通道蛋白特定位点(P266T)突变,观察对ATP敏感性钾通道电生理特性的影响。 方法: 将Kir6.2及其突变子P266T的cDNA导入人胚肾细胞，表达ATP敏感性钾通道，用膜片钳方法研究K_ATP电生理特性。 结果: K_ATP(P266T)开放能力是野生型的2倍; K_ATP(P266T)密度仅是野生型20%; K_ATP(P266T)对ATP敏感性降低;对pH敏感性增高。 结论: K_ATP特定位点(P266T)突变可改变K_ATP电生理特性。     

Prostaglandin E2 is a major soluble factor produced by stromal cells for preventing inflammatory cytokine production from dendritic cells

Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-alpha production. Using molecular-size selection and HPLC, we determined that prostaglandin E2 (PGE2) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE2 is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

ROS介导线粒体ATP敏感性钾通道开放剂对缺氧脑的保护作用

目的：观察线粒体ATP敏感性钾通道(mitoKATP)及活性氧（ROS）在缺氧脑保护中的作用及其相互关系。 方法： 采用脑片灌流及电生理学技术，细胞外记录海马CA1区的群体锋电位(PS)和缺氧去极化电位(HD)。 结果： 用mitoKATP开放剂diazoxide (300 μmol/L) 预处理海马脑片，可延长HD的潜伏期及缺氧后PS消失的时间，提高复氧后PS的恢复率。该作用可被mitoKATP阻断剂5-hydroxydecanoic acid (200 μmol/L) 所阻断。以ROS清除剂N-2-mercaptopropionyl glycine (MPG) (500 μmol/L) 预处理海马脑片，可减弱diazoxide 的作用。单独使用MPG对PS及HD无明显影响。 结论： ROS介导了mitoKATP开放剂对缺氧脑的保护作用。     

线粒体KATP通道开放剂与缺氧预适应对乳鼠窦房结细胞起搏离子流作用的比较

目的： 通过比较线粒体K_ATP通道开放剂及缺氧预适应（HP）对缺氧/复氧（H/R）乳鼠窦房结细胞起搏离子流（I_f）的作用，探讨HP对H/R时窦房结细胞电生理活动的保护机制。 方法： 取培养 2 d 的乳鼠窦房结细胞，随机分为①对照组；②H/R组；③HP组；④ diazoxide（线粒体KATP通道开放剂）+H/R组；⑤5-HD（线粒体K_ATP通道阻断剂）+HP组。以全细胞膜片钳技术检测I_f。 结果： ①H/R组各指令电压下的If密度显著高于对照组，激活曲线右移，半数最大激活电压由(-98.9±2.4)mV变为(-85.1±2.5)mV(P＜0.01)；②HP及diazoxide预处理能显著抑制I/R后升高的I_f密度，使激活曲线左移，半数最大激活电压分别为(-90.7±5.0)mV(P＜0.01)及(-92.2±1.9)mV(P＜0.01)；③5-HD预处理阻断HP效应，使I_f密度增加并使激活曲线右移，半数最大激活电压为(-86.3±2.7)mV(P＜0.01)。 结论： 线粒体K_ATP通道开放剂预处理能模拟HP效应，对抗H/R对乳鼠窦房结细胞I_f 的影响，有助于维护H/R时窦房结细胞电生理活动的相对稳定性。     

Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus delected, Ndrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on Ndrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both Ndrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of Ndrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.     

豚鼠气道平滑肌ATP敏感钾通道电流的动力学特性

气道平滑肌的主要作用是调节气道的紧张度和口径,而钾通道开放剂可经ATP敏感钾通道(ATP-sensitive potassiumchannel,KATP通道)使气道平滑肌松弛,此作用能被KATP通道特异性阻断剂优降糖阻断[1]。