Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells

The patch-clamp open-cell recording configuration has been used to investigate the effects of non-hydrolyzable analogues of ATP on the diazoxide-activation of K_ATP channels in the insulin-secreting cell line RINm5F. K⁺ channels inhibited by 0.1, 0.5 and 1.0 mM ATP were consistently activated by 200 μM diazoxide. During sustained activation of channels, exchange of ATP for either AMP-PNP, AMP-PCP or ATPγS abolished the effects of diazoxide. If diazoxide was added to the membrane in the continued presence of AMP-PNP, AMP-PCP or ATPγS either no effects were observed or alternatively a small transient activation of channels occurred. This study suggests that protein phosphorylation is necessary for diazoxide to activate ATP-sensitive potassium channels in insulin-secreting cells.     

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels

ATP-sensitive potassium channels (K_ATP) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K_ATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K_ATP channels solved by cryoelectron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.     

Late sodium current in failing heart: Friend or foe?

Most cardiac Na⁺ channels open transiently upon membrane depolarization and then are quickly inactivated. However, some channels remain active, carrying the so-called persistent or late Na⁺ current (I_NaL) throughout the action potential (AP) plateau. Experimental data and the results of numerical modeling accumulated over the past decade show the emerging importance of this late current component for the function of both normal and failing myocardium. I_NaL is produced by special gating modes of the cardiac-specific Na⁺ channel isoform. Heart failure (HF) slows channel gating and increases I_NaL, but HF-specific Na⁺ channel isoform underlying these changes has not been found. Na⁺ channels represent a multi-protein complex and its activity is determined not only by the pore-forming subunit but also by its auxiliary β subunits, cytoskeleton, calmodulin, regulatory kinases and phosphatases, and trafficking proteins. Disruption of the integrity of this protein complex may lead to alterations of I_NaL in pathological conditions. Increased I_NaL and the corresponding Na⁺ flux in failing myocardium contribute to abnormal repolarization and an increased cell Ca²⁺ load. Interventions designed to correct I_NaL rescue normal repolarization and improve Ca²⁺ handling and contractility of the failing cardiomyocytes. This review considers (1) quantitative integration of I_NaL into the established electrophysiological and Ca²⁺ regulatory mechanisms in normal and failing cardiomyocytes and (2) a new therapeutic strategy utilizing a selective inhibition of I_NaL to target both arrhythmias and impaired contractility in HF.     

Role of pacemaking current in cardiac nodes: insights from a comparative study of sinoatrial node and atrioventricular node

Cardiac pacemaking in the sinoatrial (SA) node and atrioventricular (AV) node is generated by an interplay of many ionic currents, one of which is the funny pacemaker current (I_f). To understand the functional role of I_f in two different pacemakers, comparative studies of spontaneous activity and expression of the HCN channel in mouse SA node and AV node were performed. The intrinsic cycle length (CL) is 179±2.7 ms (n=5) in SA node and 258±18.7 ms (n=5) in AV node. Blocking of I_f current by 1 μmol/L ZD7288 increased the CL to 258±18.7 ms (n=5) and 447±92.4 ms (n=5) in SA node and AV node, respectively. However, the major HCN channel, HCN4 expressed at low level in the AV node compared to the SA node. To clarify the discrepancy between the functional importance of I_f and expression level of HCN4 channel, a SA node cell model was used. Increasing the I_f conductance resulted in decreasing in the CL in the model, which explains the high pacemaking rate and high expression of HCN channel in the SA node. Resistance to the blocking of I_f in the SA node might result from compensating effects from other currents (especially voltage sensitive currents) involved in pacemaking. The computer simulation shows that the difference in the intrinsic CL could explain the difference in response to I_f blocking in these two cardiac nodes.     

Simulation of spontaneous action potentials of cardiomyocytes in pulmonary veins of rabbits

Atrial fibrillation is the most prevalent arrhythmia, but the mechanisms by which it develops are not clear. Recently, over 90% of paroxysmal atrial fibrillation was found to be located inside the main pulmonary veins (PVs). We found that single cardiac myocytes isolated from the main PVs of rabbits generate spontaneous action potentials (SAP). We therefore assayed the electrical characteristics of these cardiomyocytes. Among the diverse ionic currents identified were I_Na, I_Ca,L, I_K1, I_Kr, I_Ks, I_to, I_Ksus, I_ncx, I_pump, I_KH and I_Cl,Ca. In contrast, I_K1 was minimal, I_Ks could be detected only in the presence of 10 μM forskolin, and we were unable to detect I_f and I_Ca,T, the most important currents for pacemaking activity in sinoatrial node cells. To identify the main cause of SAP, we developed a model that can explain the electrical properties of these cardiomyocytes. After reconstructing the ionic currents based on experimental observations, we were able to use our model to successfully reconstruct the characteristics of the SAP of PV cardiomyocytes. The simulation showed that the major currents contributing to pacemaking depolarization were I_CaL, I_Kr, a background current and Na⁺–K⁺ pump current. Deactivation kinetics of I_Kr was one of the major determinants of the rate of pacemaking depolarization. The steady state inactivation of I_to was shifted to the negative voltage and the activity of I_to was minimal in the range of the SAP. The major currents for the repolarization were I_Kr and I_pump. The amplitude of most currents in these cardiac myocytes was small and no currents did not exceed 30 pA during the SAP, indicating that slight activation of other inward or outward currents will have profound effects on the SAP. To our knowledge, this report is the first to show the simulation of SAP of PV cardiomyocytes. This model may help to study on the electrophysiological basis of paroxysmal atrial fibrillation originating from PVs.     

Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassium channels

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts via both ionotropic GABA-A and metabotropic GABA-B receptors. We evaluated the interaction of receptors with members of the inwardly rectifying potassium (Kir 3) channel family, which also play an important role in neuronal transmission and membrane excitability. These channels are functionally regulated by GABA-B receptors. Possible physical interactions between GABA-B receptor and Kir 3 channels expressed in HEK cells were evaluated using Bioluminescence Resonance Energy Transfer (BRET) experiments, co-immunoprecipitation and confocal microscopy. Our data indicate that Kir 3 channels and Gβγ subunits can interact with the GABA-B₁ subunits independently of the GABA-B₂ subunit or Kir 3.4 which are ultimately responsible for their targetting to the cell surface. Thus signalling complexes containing GABA-B receptors, G proteins and Kir channels are formed shortly after biosynthesis most likely in the endoplasmic reticulum.     

Protective effects of the potassium channel opener-diazoxide against injury in neonatal rat ventricular myocytes

The mitochondrial ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. Similarly to plasma membrane K(ATP), the mitochondrial channel is inhibited by antidiabetic sulfonylureas and activated by potassium channel openers, such as diazoxide. In the present work, the cytoprotective properties of diazoxide on the H9c2 cardiac myoblast cell line and neonatal rat ventricular cardiomyocytes were analysed. It was observed that 100 micromol/l diazoxide protected neonatal rat ventricular cardiomyocytes, but not H9c2 myoblasts, against injury induced by hydrogen peroxide or simulated ischemia. Moreover, diazoxide prevented hydrogen peroxide-induced mitochondrial potential depolarisation in neonatal rat ventricular cardiomyocytes. Diazoxide, at the same time, did not affect the expression level of the anti-apoptotic protein bcl-2 in these cells. The protective effects of diazoxide were suppressed by 5-hydroxydecanoic acid, a potassium channel blocker. These observations suggest that activation of the mitochondrial ATP-regulated potassium channel plays an important role in protection of neonatal cardiomyocytes against injury.     

Dihydropyridine KATP potassium channel openers

Three related series of dihydropyridine K_ATP potassium channel openers are described.     

Electrophysiological modelling of pulmonary artery smooth muscle cells in the rabbits--special consideration to the generation of hypoxic pulmonary vasoconstriction

In vascular smooth muscle cells, it has been suggested that membrane potential is an important component that initiates contraction. We developed a mathematical model to elucidate the quantitative contributions of major ion currents [a voltage-gated L-type Ca²⁺ current (I_CaL), a voltage-sensitive K⁺ current (I_KV), a Ca²⁺-activated K⁺ current (I_KCa) and a nonselective cation current (I_NSC)] to membrane potential. In order to typify the diverse nature of pulmonary artery smooth muscle cells (PASMCs), we introduced parameters that are not fixed (variable parameters). The population of cells with different parameters was constructed and the cells that have the electrophysiological properties of PASMCs were selected. The contributions of each membrane current were investigated by sensitivity analysis and modification of the current parameters. Consequently, I_KV and I_NSC were found to be the most important currents that affect the membrane potential. The occurrence of depolarisation in hypoxic pulmonary vasoconstriction (HPV) was also examined. In hypoxia, I_KV and I_KCa were reduced, but the consequent depolarisation in simulation was not enough to initiate contractions. If we add an increase of I_NSC (2.5-fold), the calculated membrane potential was enough to induce contraction. From the results, we conclude that the balance of various ion channel activities determines the resting membrane potential of PASMCs and our model was successful in explaining the depolarisation in HPV. Therefore, this model can be a powerful tool to investigate the various electrical properties of PASMCs in both normal and pathological conditions.     

Cell-type specific depression of neuronal excitability in rat hippocampus by activation of ATP-sensitive potassium channels

The contribution of ATP-sensitive potassium (K(ATP)) channels to neuronal excitability was studied in different types of pyramidal cells and interneurones in hippocampal slices prepared from 9- to 15-day-old rats. The presence of functional K(ATP) channels in the neurones was detected through the sensitivity of whole-cell currents to diazoxide, a K(ATP) channel opener, and to tolbutamide, a K(ATP) channel inhibitor. The percentages of neurones with K(ATP) channels increase in the sequence: CA1 pyramidal cells (37%)相似文献   

'Ca(2+)-induced Ca(2+) entry' or how the L-type Ca(2+) channel remodels its own signalling pathway in cardiac cells

The adjustment of Ca²⁺ entry in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body. In this review, we present the concept that Ca²⁺ can promote its own entry through Ca²⁺ channels by different mechanisms. We refer to it under the general term of ‘Ca²⁺-induced Ca²⁺ entry’ (CICE). We review short-term mechanisms (usually termed facilitation) that involve a stimulating effect of Ca²⁺ on the L-type Ca²⁺ current (I_Ca-L) amplitude (positive staircase) or a lessening of Ca²⁺-dependent inactivation of I_Ca-L. This latter effect is related to the amount of Ca²⁺ released by ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR). Both effects are involved in the control of action potential (AP) duration. We also describe a long-term mechanism based on Ca²⁺-dependent down-regulation of the Kv4.2 gene controlling functional expression of the repolarizing transient outward K⁺ current (I_to) and, thereby, AP duration. This mechanism, which might occur very early during the onset of hypertrophy, enhances Ca²⁺ entry by maintaining Ca²⁺ channel activation during prolonged AP. Both Ca²⁺-dependent facilitation and Ca²⁺-dependent down-regulation of I_to expression favour AP prolongation and, thereby, promote sustained voltage-gated Ca²⁺ entry used to enhance excitation–contraction (EC) coupling (with no change in the density of Ca²⁺ channels per se). These self-maintaining mechanisms of Ca²⁺ entry have significant functions in remodelling Ca²⁺ signalling during the cardiac AP. They might support a prominent role of Ca²⁺ channels in the establishment and progression of abnormal Ca²⁺ signalling during cardiac hypertrophy and congestive heart failure.     

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

硫化氢对家兔窦房结起搏细胞的电生理效应

本研究应用细胞内微电极技术,观察硫化氢(hydrogen sulfide,H2S)对家兔窦房结起搏细胞的电生理效应.结果表明:(1)NaHs(H2S供体)50、100、200 μmol/L浓度依赖地降低家兔窦房结起搏细胞4相去极化速率及起搏放电频率.(2)ATP敏感性钾(ATP-sensitive K ,KATP)通道阻断剂格列苯脲(glybenclamide,Gli,20 μmol/L)阻断NariS(100 μmol/L)的电生理效应.(3)预先应用起搏离子流(pacemaker currenL,If)通道阻断剂氯化铯(CsCl,2 mmol/L)对Naris(100μmol/L.)的电生理效应无影响.(4)胱硫醚-γ裂解酶(cystathionine γ-lyase,CSE)的不可逆抑制剂DL-propargylglycine (PPG,200 μmol/L)的家兔窦房结起搏细胞的动作电位参数无影响.以上结果提示,H2S对家兔窦房结起搏细胞有负性变时作用,这些效应可能与其开放KATP通道,增加K 外流有关,与If无关.本实验没有发现窦房结起搏细胞内有CSE催化产生的内源性H2S的合成.     

Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f)

The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.     

激活SUR2B/Kir6.1通道扩张肺微动脉的分子机制

目的：以ATP敏感性钾通道（K_ATP） SUR2B/Kir6.1亚型开放剂埃他卡林（Ipt）为工具药,研究激活SUR2B/Kir6.1通道扩张肺微动脉作用特征,并探讨其可能的分子机制。方法：利用离体微血管压力-直径监测灌流技术,检测Ipt对大鼠四级肺微动脉的舒张效应（n=6~8）,观察内皮损伤后或用K_ATP通道拮抗剂格列苯脲（Gli）、环氧合酶（COX）抑制剂吲哚美辛（Indo）、一氧化氮合酶（NOS）抑制剂L-Nω-硝基精氨酸甲酯（L-NAME）预孵后肺微动脉舒张率的变化。结果：Ipt能够扩张肺微动脉,最大舒张率为（60.53±2.08）%。内皮细胞损伤后,Ipt扩张肺微动脉作用明显减弱,最大舒张率为（9.47±1.56）%,与对照组相比存在显著性差异（P<0.01）。预孵Gli、Indo、L-NAME后,最大舒张率分别下降为（17.49±1.47）%、（37.00±3.88）%、（24.91±2.30）%,与对照组相比均存在显著性差异（P<0.01）。结论：其选择性开放K_ATP通道SUR2B/Kir6.1扩张肺微动脉作用具有内皮细胞依赖性,与其促进内皮细胞释放一氧化氮（NO）和前列环素（PGI₂）相关。     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

Vasopressin directly closes ATP-sensitive potassium channels evoking membrane depolarization and an increase in the free intracellular Ca2+ concentration in insulin-secreting cells.

The effects of arginine-vasopressin (AVP) (0.01-1 microM) on membrane potential, [Ca2+]i and ATP-sensitive potassium channels have been studied in the insulin-secreting cell line RINm5F. In whole cells, with an average spontaneous cellular transmembrane potential of -64 +/- 3 mV (n = 33) and an average basal [Ca2+]i of 102 +/- 6 nM (n = 40), AVP evoked: (i) membrane depolarization, (ii) voltage-dependent Ca2+ spike-potentials and (iii) a sharp rise in [Ca2+]i. Single-channel current events recorded from excised outside-out membrane patches show that AVP closes potassium channels that are also closed by tolbutamide (100 microM) and opened by diazoxide (100 microM). AVP acts on KATP channels specifically from the outside of the membrane and a soluble cytosolic messenger appears not to be involved, since there is no channel activation in cell-attached membrane patches when the peptide is added to the bath solution.