Kir6.2-containing ATP-sensitive potassium channels protect cortical neurons from ischemic/anoxic injury in vitro and in vivo

ATP-sensitive potassium (K(ATP)) channels are weak inward rectifiers that appear to play an important role in protecting neurons against ischemic damage. Cerebral stroke is a major health issue, and vulnerability to stroke damage is regional within the brain. Thus, we set out to determine whether K(ATP) channels protect cortical neurons against ischemic insults. Experiments were performed using Kir6.2(-/-) K(ATP) channel knockout and Kir6.2(+/+) wildtype mice. We compared results obtained in Kir6.2(-/-) and wildtype mice to evaluate the protective role of K(ATP) channels against focal ischemia in vivo, and, using cortical slices, against anoxic stress in vitro. Immunohistochemistry confirmed the presence of K(ATP) channels in the cortex of wildtype, but not Kir6.2(-/-), mice. Results from in vivo and in vitro experimental models indicate that Kir6.2-containing K(ATP) channels in the cortex provide protection from neuronal death. Briefly, in vivo focal ischemia (15 min) induced severe neurological deficits and large cortical infarcts in Kir6.2(-/-) mice, but not in wildtype mice. Imaging analyses of cortical slices exposed briefly to oxygen and glucose deprivation (OGD) revealed a substantial number of damaged cells (propidium iodide-labeled) in the Kir6.2(-/-) OGD group, but few degenerating neurons in the wildtype OGD group, or in the wildtype and Kir6.2(-/-) control groups. Slices from the three control groups had far more surviving cells (anti-NeuN antibody-labeled) than slices from the Kir6.2(-/-) OGD group. These findings suggest that stimulation of endogenous cortical K(ATP) channels may provide a useful strategy for limiting the damage that results from cerebral ischemic stroke.     

ATP敏感性钾通道亚基Kir6.1、Kir6.2在MPTP诱导帕金森病小鼠纹状体和黑质内的改变

为探讨ATP敏感性钾通道(KATP)亚基Kir6.1、Kir6.2在帕金森病(PD)病理生理机制中的可能作用。本研究采用蛋白免疫印迹分析(Western blot)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的PD小鼠模型黑质、纹状体Kir6.1、Kir6.2在不同时间点表达变化进行检测,并与酪氨酸羟化酶(TH)的变化进行比较。结果发现:(1)与正常对照组相比,黑质、纹状体TH蛋白的表达在给药后第1d即开始下降,且呈时间依赖性下降(P<0.01);(2)黑质Kir6.1蛋白的表达在给药后第5d才开始下降(P<0.01);而纹状体Kir6.1蛋白的表达在给药后第5d才开始升高(P<0.01);(3)黑质Kir6.2蛋白的表达在给药后第5d才开始明显升高(P<0.01);而纹状体Kir6.2蛋白的表达在给药后第3d轻度升高(P<0.05),第5d又明显降低(P<0.01)。以上结果提示作为KATP通道亚基的Kir6.1、Kir6.2在MPTP的作用下,可能通过参与星形胶质细胞的活化、胆碱能突触传递的抑制以及自身代偿和修复在PD的病理生理过程中发挥了重要的角色。     

ATP-sensitive K+ channels in rat colonic epithelium

ATP-sensitive K⁺ (K_ATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that K_ATP channels contribute to epithelial K⁺ currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I _sc) induced by the K_ATP channel opener pinacidil (5 10^?4?mol?l^?1) were measured in Ussing chambers under control conditions and in the presence of different blockers of K_ATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K⁺ channel opener, when administered at the serosal side, induced an increase in I _sc consistent with the induction of transepithelial Cl^? secretion after activation of basolateral K⁺ channels, whereas mucosal administration of pinacidil resulted in a negative I _sc. The increase in I _sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10^?4?mol l^?1) and gliclazide (10^?6?mol l^?1), but was resistant even against a high concentration (10^?2?mol l^?1) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I _sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl^? currents across apical Cl^? channels in basolaterally depolarized epithelia indicating that the negative I _sc induced by mucosal pinacidil is due to a transient inhibition of Cl^? secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, K_ATP channels are present in the basolateral membrane of the colonic epithelium; their exact subunit composition, however, has still to be revealed.     

Role of sarcolemmal ATP-sensitive K+ channels in the regulation of sinoatrial node automaticity: an evaluation using Kir6.2-deficient mice

The role of cardiac sarcolemmal ATP-sensitive K⁺ (K_ATP) channels in the regulation of sinoatrial node (SAN) automaticity is not well defined. Using mice with homozygous knockout (KO) of the Kir6.2 (a pore-forming subunit of cardiac K_ATP channel) gene, we investigated the pathophysiological role of K_ATP channels in SAN cells during hypoxia. Langendorff-perfused mouse hearts were exposed to hypoxic and glucose-free conditions (hypoxia). After 5 min of hypoxia, sinus cycle length (CL) was prolonged from 207 ± 10 to 613 ± 84 ms ( P < 0.001) in wild-type (WT) hearts. In Kir6.2 KO hearts, CL was slightly prolonged from 198 ± 17 to 265 ± 32 ms. The CL of spontaneous action potentials of WT SAN cells, recorded in the current-clamp mode, was markedly prolonged from 410 ± 56 to 605 ± 108 ms ( n = 6, P < 0.05) with a decrease of the slope of the diastolic depolarization (SDD) after the application of the K⁺ channel opener pinacidil (100 μ m ). Pinacidil induced a glibenclamide (1 μ m )-sensitive outward current, which was recorded in the voltage-clamp mode, only in WT SAN cells. During metabolic inhibition by 2,4-dinitrophenol, CL was prolonged from 292 ± 38 to 585 ± 91 ms ( P < 0.05) with a decrease of SDD in WT SAN cells but not in Kir6.2 KO SAN cells. Diastolic Ca²⁺ concentration, measured by fluo-3 fluorescence, was decreased in WT SAN cells but increased in Kir6.2 KO SAN cells after short-term metabolic inhibition. In conclusion, the present study using Kir6.2 KO mice indicates that, during hypoxia, activation of sarcolemmal K_ATP channels in SAN cells inhibits SAN automaticity, which is important for the protection of SAN cells.     

Protein kinase C modulation of recombinant ATP-sensitive K+ channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B

The molecular identity of smooth muscle ATP-sensitive K⁺ channels (K_ATP) is not established with certainty. Patch clamp methods were employed to determine if recombinant K_ATP channels composed of Kir6.1 and SUR2B subunits expressed by human embryonic kidney (HEK293) cells share an identical modulation by protein kinase C (PKC) with the vascular K_NDP subtype of K_ATP channel. The open probability of Kir6.1/SUR2B channels was determined before and after sequential exposure to pinacidil (50 μM) and the combination of pinacidil and phorbol 12,13-dibutyrate (PdBu; 50 n m ). Treatment with PdBu caused a decline in channel activity, but this was not seen with an inactive phorbol ester, 4α-phorbol 12,13-didecanoate (PdDe; 50 n m ). Angiotensin II (0.1 μM) induced a similar inhibition of Kir6.1/SUR2B channels in cells expressing angiotensin AT₁ receptors. The effects of PdBu and angiotensin II were blocked by the PKC inhibitor, chelerythrine (3 μM). Purified PKC inhibited Kir6.1/SUR2B activity (in 0.5 m m ATP/ 0.5 m m ADP), and the inhibition was blocked by a specific peptide inhibitor of PKC, PKC(19-31). In contrast, PdBu increased the activity of recombinant K_ATP channels composed of Kir6.2 and SUR2B, or the combination of Kir6.1, Kir6.2 and SUR2B subunits. The results indicate that the modulation by PKC of Kir6.1/SUR2B, but not Kir6.2/SUR2B or Kir6.1-Kir6.2/SUR2B channel gating mimics that of native vascular K_NDP channels. Physiological inhibition of vascular K_ATP current by vasoconstrictors which utilize intracellular signalling cascades involving PKC is concluded to involve the modulation of K_NDP channel complexes composed of four Kir6.1 and their associated SUR2B subunits.     

ATP敏感钾通道在心血管系统中的作用

1983年,Noma[1]首次报道细胞内ATP可抑制豚鼠心室肌选择性钾通道(即KATP),并推测KATP激活与心肌缺血时动作电位缩短有关,KATP激活可降低细胞内 ATP的消耗,避免细胞发生不可逆性损伤.近年来大量研究表明KATP在心血管系统的病理生理过程中具有重要作用.     

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敏感性钾通道突变与新生儿糖尿病iDEND综合征

ATP敏感性钾通道(ATP-sensitive K+chan-nels,KATP)由SUR1和Kir6.2亚基组成,是葡萄糖刺激胰岛β细胞分泌胰岛素的关键部位。新生儿糖尿病iDEND综合征(intermediate developmental delay,epilepsy,and neonatal diabetes syndrome)是由KATP通     

ATP-independent anoxic activation of ATP-sensitive K+ channels in dorsal vagal neurons of juvenile mice in situ.

The role of ATP in anoxic activation of ATP-sensitive K+ (KATP) channels was studied in dorsal vagal neurons of mouse brainstem slices. In the whole-cell configuration, cyanide-induced chemical anoxia evoked within 10 s a 300-pA outward current that gave rise to a hyperpolarization of 24 mV. These responses were mimicked by nitrogen-aerated saline, rotenone or diazoxide and abolished by tolbutamide. The cyanide-induced hyperpolarization was due to activation of 70 pS K(ATP) channels that were half-maximally blocked by 5 microM internal ATP. Dialyzing the cells with either 1, 20 or 0 mM ATP did not, however, affect the time to onset, the kinetics or the magnitude of the cyanide-induced hyperpolarization. Impairment of ATP consumption by ouabain, vanadate or reduced temperature had no effect either. Thus, anoxia-induced activation of these KATP channels cannot be explained by a fall of cellular ATP or a concomitant rise of ADP. Anoxia-related changes of the actin cytoskeleton or the composition of the plasma membrane are also not likely to be involved, as cytochalasin D did not affect the cyanide-evoked hyperpolarization and phosphatidylinositol 4,5-bisphosphate failed to decrease the ATP sensitivity of single KATP channels. Finally, because of a lack of effects of reduced/oxidized glutathione and the oxidase blocker diphenyliodonium on the cyanide-induced hyperpolarization, cellular redox state does not appear to be involved.Our results indicate that despite a high sensitivity to ATP in excised patches, anoxic activation of KATP channels is independent of cellular ATP. Rather the ATP block seems to be removed as a consequence of impaired mitochondrial function.     

Suppression of oxidative neuronal damage after transient middle cerebral artery occlusion in mice lacking interleukin-1

Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clear this central issue, mice that were gene deficient in IL-1alpha and beta (IL-1 KO) and wild-type mice were subjected to 1-h transient middle cerebral artery occlusion (tMCAO). Expression levels of IL-1beta and IL-1 receptor I (IL-1RI) were then examined. Generation of peroxynitrite and the expression of mRNAs for nitric oxide synthase (NOS) subtypes were also determined. Immunostaining for IL-1beta was increased from 6 h and peaked at 24 h after tMCAO in the microglia and macrophage. The immunoreactivities of IL-1RI were increased progressively in the microvasculature and neuron-like cells of the ipsilateral hemisphere. Infarct volumes were significantly lower in IL-1 KO mice compared with wild-type mice 48 h after tMCAO (P<0.01). The immunoreactivities of 3-nitro-L-tyrosine were determined in the neurons and microvasculature 24 h after tMCAO and were significantly decreased in the IL-1 KO mice compared to wild-type mice. In addition, expression levels of NOS mRNA in IL-1 KO mice were lower than that measured in wild-type mice. These results indicate that IL-1 is up-regulated and may play a role in neurodegeneration by peroxynitrite production during ischemia.     

Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive K+ channels

Intracellular recordings were made from neurones located in the ventromedial hypothalamic nucleus (VMHN) of slices from rat hypothalamus. These neurones were hyperpolarized on removal of extracellular glucose, resulting in an inhibition of firing, actions which were reversed on the re-introduction of glucose. No reversal of the inhibition of firing was observed when 10 mM mannoheptulose an inhibitor of glucose metabolism, was present in addition to glucose. Increasing the mannoheptulose concentration to 20 mM resulted in further hyperpolarization. Cell-attached recordings from isolated neurones revealed that an increase in extracellular glucose inhibited a K⁺ channel and increased action current activity. ATP induced closure of this K⁺ channel when applied to inside-out membrane patches. Closure was also induced by Mg-free ATP or the nonhydrolysable ATP-analogue, adenylylimidodiphosphate indicating no requirement for ATP metabolism. We suggest that the closure of ATP-sensitive potassium channels underlies increased hypothalamic firing following an increase in extracellular glucose.     

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.     

Effects of intracellular pH on ATP-sensitive K+ channels in mouse pancreatic beta-cells.

1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells.     

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxy-benzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxy-acetophenone, on the adenosine-5-triphosphate(ATP)-sensitive K⁺ channel current (I _K.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5–1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of I _K.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K⁺ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as I _K.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on I _K.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of I _K.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP. These results suggest that these compounds: (a) modulate I _K.ATP perhaps through an intracellular mechanism, (b) bind covalently to proteins to form a Schiff base which may by responsible for the effects, and (c) may require an ADP-dependent process.     

大鼠细动脉平滑肌细胞内酸中毒对ATP敏感钾通道的影响

目的：研究细胞内酸中毒对细动脉平滑肌细胞膜ＫＡＴＰ通道的影响。方法：应用膜片钳技术的内面向外式记录酶性分离的细动脉平滑肌上ＫＡＴＰ通道。结果；当细胞内无ＡＴＰ时，细胞内酸中毒降低通道电导，通道平均开放时间和开放时间长成分有所增大，对通道开放概率无明显影响；     

Activators of ATP-sensitive K+ channels reduce anoxic depolarization in CA3 hippocampal neurons

In CA3 hippocampal neurons of the rat, brief anoxic episodes produce a depolarization which is probably due to a synaptic release of glutamate. Diazoxide, an activator of ATP-sensitive K+ channels (K+ ATP), blocks the anoxic depolarization and has no effect in control oxygenated artificial cerebrospinal fluid. The hormone somatostatin which activates K+ ATP channels in the pancreas also reduces the anoxic depolarization in CA3 neurons. We suggest that drugs that open K+ ATP channels may constitute a novel approach to selectivity reducing the deleterious effects of excessive release of glutamate during anoxia without producing a generalized blockade of glutamatergic synaptic transmission.