Absence of ischemic preconditioning protection in diabetic sheep hearts: Role of sarcolemmal KATP channel dysfunction

Sarcolemmal ATP-sensitive potassium (KATP) channels have been mentioned to participate in preconditioning protection. Since these channels are altered in diabetes, it would be possible that preconditioning does not develop in diabetic (D) hearts. The purpose of this study was to assess whether early (EP) and late (LP) ischemic preconditioning protect diabetic hearts against stunning in a conscious diabetic sheep model and whether diabetes might have altered KATP channel functioning. Sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control (DC, 12 min of ischemia (I) followed by 2 h of reperfusion (R)), early preconditioning (DEP, six 5 min I – 5 min R periods were performed before the 12 min I) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min I). Regional mechanics during reperfusion was evaluated as the percent recovery of wall thickening fraction (%WTH) expressed as percentage of basal values (100%) and KATP behaviour was indirectly assessed by monophasic action potential duration (MAPD) and sensitivity to glibenclamide blockade (0.1 and 0.4 mg/Kg). The results were compared to those obtained in normal (N) sheep. EP and LP protected against stunning in normal sheep (%WTH: NC = 63 ± 3.7, NLP = 80 ± 5**, NEP equals; 78 ± 3*, *p < 0.05 and **p < 0.01 against NC) whereas contrary results occurred in diabetic ones, where DLP (%WTH = 60 ± 4) afforded a similar recovery to DC (%WTH = 54 ± 5) and DEP surprisingly worsened instead of improving mechanical function (%WTH = 38 ± 6, p < 0.01 against DC). KATP channel behaviour appeared altered in diabetic hearts as shown by MAPD during ischemia in normal sheep (153 ± 9 msec) compared to diabetic ones (128 ± 11 msec, p < 0.05) and by the sensitivity to glibenclamide (while 0.4 mg/Kg blocked action potential shortening in normal and diabetic animals, 0.1 mg/Kg completely blocked KATP in diabetic but not in normal hearts, p ( 0.05). A sarcolemmal KATP channel dysfunction might afford a primary approach to explain the absence of ischemic preconditioning protection against stunning in diabetic sheep.     

葛根素对豚鼠心室肌细胞钾离子通道的影响

目的 :观察葛根素对豚鼠单个心室肌细胞钾离子通道的影响。方法 :采用内面向外膜片钳单通道记录技术。结果 :葛根素 2 0 μmol/L ,4 0 μmol/L ,80 μmol/L对单个心肌细胞钾离子通道的开放概率 (P0 )有抑制作用 ,在 80μmol/L时 ,P0 值从 0 .86 7± 0 .13降至 0 .0 19± 0 .0 1,与用药前比较有显著差异 (n =5 ,P <0 .0 1)。结论 :葛根素能抑制心肌细胞钾离子通道是其抗心律失常的分子机制     

Effect of sodium channel abundance on Drosophila development,reproductive capacity and aging

The voltage-gated Na⁺ channels (VGSC) are complex membrane proteins responsible for generation and propagation of the electrical signals through the brain, the skeletal muscle and the heart. The levels of sodium channels affect behavior and physical activity. This is illustrated by the maleless mutant allele (mle^napts) in Drosophila, where the decreased levels of voltage-gated Na⁺ channels cause temperature-sensitive paralysis.

Here, we report that mle^napts mutant flies exhibit developmental lethality, decreased fecundity and increased neurodegeneration. The negative effect of decreased levels of Na⁺ channels on development and ts-paralysis was more pronounced at 18 and 29°C than at 25°C, suggesting particular sensitivity of the mle^napts flies to temperatures above and below normal environmental conditions. Similarly, longevity of mle^napts flies was unexpectedly short at 18 and 29°C compared with flies heterozygous for the mle^napts mutation. Developmental lethality and neurodegeneration of mle^napts flies was partially rescued by increasing the dosage of para, confirming a vital role of Na⁺ channels in development, longevity and neurodegeneration of flies and their adaptation to temperatures.     

Effect of sodium channel abundance on Drosophila development, reproductive capacity and aging

The voltage-gated Na (+) channels (VGSC) are complex membrane proteins responsible for generation and propagation of the electrical signals through the brain, the skeletal muscle and the heart. The levels of sodium channels affect behavior and physical activity. This is illustrated by the maleless mutant allele (mle (napts)) in Drosophila, where the decreased levels of voltage-gated Na(+) channels cause temperature-sensitive paralysis. Here, we report that mle (napts) mutant flies exhibit developmental lethality, decreased fecundity and increased neurodegeneration. The negative effect of decreased levels of Na(+) channels on development and ts-paralysis was more pronounced at 18 and 29°C than at 25°C, suggesting particular sensitivity of the mle (napts) flies to temperatures above and below normal environmental conditions. Similarly, longevity of mle (napts) flies was unexpectedly short at 18 and 29°C compared with flies heterozygous for the mle (napts) mutation. Developmental lethality and neurodegeneration of mle (napts) flies was partially rescued by increasing the dosage of para, confirming a vital role of Na(+) channels in development, longevity and neurodegeneration of flies and their adaptation to temperatures.     

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca²⁺ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca²⁺ channel blocker diltiazem. Percent wall thickening fraction (% WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 g/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca²⁺ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca²⁺ influx.     

KCa3.1 upregulation preserves endothelium‐dependent vasorelaxation during aging and oxidative stress

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium‐dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial K_Ca3.1, which contributes to EDR, is upregulated by H₂O₂. We investigated whether K_Ca3.1 upregulation compensates for diminished EDR to NO during aging‐related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1‐phosphate were increased in aged wild‐type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild‐type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age‐matched wild‐type mice. Increased H₂O₂ levels induced Fyn and extracellular signal‐regulated kinases (ERKs) phosphorylation and K_Ca3.1 upregulation. Catalase/GPX1 double knockout (catalase^?/?/GPX1^?/?) upregulated K_Ca3.1 in MAECs. NO production was decreased in aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? MAECs. However, K_Ca3.1 activation‐induced, N^G‐nitro‐l ‐arginine‐, and indomethacin‐resistant EDR was increased without a change in acetylcholine‐induced EDR in aortic rings from aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1‐phosphate induced similar changes in levels of the antioxidant enzymes and upregulated K_Ca3.1. Our findings suggest that, during aging‐related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H₂O₂ and thereby upregulate K_Ca3.1 expression and function via a H₂O₂/Fyn‐mediated pathway. Altogether, enhanced K_Ca3.1 activity may compensate for decreased NO signaling during vascular aging.     

Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N- and C-termini regions of the ROMK1 K+ channel.

The hydropathy plot of the inwardly rectifying ROMK1 K+ channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post-M2). Peptides that correspond to M0, post-M2, and a control peptide, pre-M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self-assemble and coassemble in the membrane-bound state. CD spectroscopy revealed that both M0 and post-M2 adopt highly alpha-helical structures in 1% SDS and 40% TFE/water, whereas pre-M0 is not alpha-helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD-labeled peptides demonstrated that both M0 and post-M2, but not pre-M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10(3)-10(5) M-1. A surface localization for both post-M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/ acceptor (rhodamine) peptides revealed that M0 and post-M2 can coassemble in their membrane-bound state, but cannot self-associate when membrane-bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K+ channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994 Science 264:844-847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039-1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane-bound polypeptides are also discussed.     

Alterations in intrinsic neuronal excitability during normal aging

Normal aging subjects, including humans, have difficulty learning hippocampus-dependent tasks. For example, at least 50% of normal aging rabbits and rats fail to meet a learning criterion in trace eyeblink conditioning. Many factors may contribute to this age-related learning impairment. An important cause is the reduced intrinsic excitability observed in hippocampal pyramidal neurons from normal aging subjects, as reflected by an enlarged postburst afterhyperpolarization (AHP) and an increased spike-frequency adaptation (accommodation). In this review, we will focus on the alterations in the AHP and accommodation during learning and normal aging. We propose that age-related increases in the postburst AHP and accommodation in hippocampal pyramidal neurons play an integral role in the learning impairment observed in normal aging subjects.     

Disrupting KATP channels diminishes the estrogen-mediated protection in female mutant mice during ischemia-reperfusion

Background

Estrogen has been shown to mediate protection in female hearts against ischemia-reperfusion (I-R) stress. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, cardiac ATP-sensitive potassium channels (KATP) remain quiescent under normal physiological conditions but they are activated by stress stimuli to confer protection to the heart. It remains unclear whether KATP is a regulatory target of estrogen in the female-specific I-R signaling pathway. In this study, we aimed at delineating the molecular mechanism underlying estrogen modulation on KATP channel activity during I-R.

Materials and methods

We employed KATP knockout mice in which SUR2 is disrupted (SUR2KO) to characterize their I-R response using an in vivo occlusion model. To test the protective effects of estrogen, female mice were ovariectomized and implanted with 17β-estradiol (E2) or placebo pellets (0.1 μg/g/day, 21-day release) before receiving an I-R treatment. Comparative proteomic analyses were performed to assess pathway-level alterations between KO-IR and WT-IR hearts.

Results and discussion

Echocardiographic results indicated that KO females were pre-disposed to cardiac dysfunction at baseline. The mutant mice were more susceptible to I-R stress by having bigger infarcts (46%) than WT controls (31%). The observation was confirmed using ovariectomized mice implanted with E2 or placebo. However, the estrogen-mediated protection was diminished in KO hearts. Expression studies showed that the SUR2 protein level, but not RNA level, was up-regulated in WT-IR mice relative to untreated controls possibly via PTMs. Our antibodies detected different glycosylated SUR2 receptor species after the PNGase F treatment, suggesting that SUR2 could be modified by N-glycosylation. We subsequently showed that E2 could further induce the formation of complex-glycosylated SUR2. Additional time-point experiments revealed that I-R hearts had increased levels of N-glycosylated SUR2; and DPM1, the first committed step enzyme in the N-glycosylation pathway. Comparative proteomic profiling identified 41 differentially altered protein hits between KO-IR and WT-IR mice encompassing those related to estrogen biosynthesis.

Conclusions

Our findings suggest that KATP is likely a downstream regulatory target of estrogen and it is indispensable in female I-R signaling. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be effective to enhance KATP channel subunit expression in I-R.     

Five-group distribution of the Shaker-like K+ channel family in higher plants

Abstract In higher plants, potassium channels of the Shaker family have been shown to play crucial roles in the uptake of K⁺ from the soil solution and subsequent transport of this ion at the cell, tissue, and organ levels. In the model plant Arabidopsis thaliana, this family is composed of nine members, which are the best characterized among plant channels at the protein, gene, and functional property levels. Plant Shaker channels share a common structure: a hydrophobic core composed of six transmembrane segments, a long cytoplasmic C-terminal region harboring a putative cyclic nucleotide binding domain, and a K_HA domain. Many channels also contain an ankyrin domain between the putative cyclic nucleotide binding domain and the K_HA domain. The analysis of 44 Shaker channels from plants revealed a five-group classification. The members of each group share high sequence and structure similarities. This grouping also correlates with the diversification of the functional properties of the proteins, as members of an individual group have roughly the same electrophysiological characteristics. Analysis of the intron positions showed that the gene structures are also quite well conserved within the five groups. A correlation linking the evolution of the sequences and the positioning of the introns was established. Finally, a moss sequence provided additional clues about the hypothetical structure of an ancestor of the present channels and suggested that the diversification of plant Shaker channels happened before the separation of monocots and dicots and after the separation of bryophytes and tracheophytes.     

Intrinsic and extrinsic determinants of ion channel localization in neurons

Neurons are an extremely diverse group of excitable cells with a wide variety of morphologies including complex dendritic trees and very long axons. The electrical properties of neurons depend not only on the types of ion channels and receptors expressed, but also on where these channels are located in the cell. Two extreme examples that illustrate the subcellular polarized nature of neurons and the tight regulation of ion channel localization can be seen at the axon initial segment and the node of Ranvier. The axon initial segment is important for initiation of action potentials in the axon, whereas the node of Ranvier is required for the rapid, faithful and efficient propagation of action potentials along the axon. Given the similarity of their functions it is not surprising that nearly every protein component of the axon initial segment is also found at the node. However, there is one very important difference between these two sites: nodes require extrinsic, glial-derived factors in order to form, whereas the axon initial segment is intrinsically determined by the neuron. This mini-review discusses recent results that have begun to clarify the intrinsic and extrinsic mechanisms underlying formation of nodes and axon initial segments, and poses several important unanswered questions regarding their unique mechanisms of formation.     

Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via KCa1.1 upregulation

K_Ca1.1 regulates smooth muscle contractility by modulating membrane potential, and age‐associated changes in K_Ca1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K_Ca1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2‐null mice. The levels of C16‐ and C18‐ceramides, sphinganine, sphingosine, and sphingosine 1‐phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild‐type and young CerS2‐null mice. The altered SL composition upregulated K_Ca1.1 and increased K_Ca1.1 currents, while no change was observed in K_Ca1.1 channel activity. The upregulation of K_Ca1.1 impaired intracellular Ca²⁺ mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3‐kinase, protein kinase C_ζ, c‐Jun N‐terminal kinases, and nuclear factor kappa‐B were found to be involved in K_Ca1.1 upregulation. Our findings suggest that age‐associated changes in SL composition or CerS2 ablation upregulate K_Ca1.1 via the phosphoinositide 3‐kinase/protein kinase C_ζ/c‐Jun N‐terminal kinases/nuclear factor kappa‐B‐mediated pathway and impair Ca²⁺ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2‐null mice exhibited similar effects to aged wild‐type mice; therefore, CerS2‐null mouse models may be utilized for investigating the pathogenesis of aging‐associated motility disorders.     

钾离子通道蛋白Shaker对果蝇心脏衰老的保护作用

钾离子通道在心肌细胞动作电位复极过程中起着重要作用。钾离子通道蛋白种类繁多,已知钾离子通道蛋白KCNQ和HERG/eag参与心脏动作电位的形成,调节心脏收缩节律。钾离子通道蛋白Shaker是果蝇(Drosophila)体内发现的第一个电压门控钾离子通道,维持神经元和肌肉细胞的电兴奋性,但是目前其在成人心脏功能中的作用仍不清楚。本研究以果蝇为模型,高频电刺激模拟心脏应激状态,观察钾离子通道蛋白shaker基因突变体的心衰发生率。同时,利用心脏特异性启动子hand4.2Gal4特异性敲低钾离子通道蛋白Shaker的表达;果蝇成体心脏生理学功能分析系统分析了1、3、5周龄特异性敲低钾离子通道蛋白Shaker的心脏表型。结果表明,shaker基因突变将严重影响果蝇心脏抗应激能力,表现在高频电刺激后的心力衰竭发生率显著性升高;心脏特异性敲低shaker基因导致5周龄果蝇心律失常发生率显著性增加;心脏特异性敲低HDAC3将显著降低果蝇寿命。综上所述,本研究推测钾离子通道蛋白Shaker在衰老过程中维护果蝇正常的心脏功能。     

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

不同烤烟基因型钾离子通道特性研究

通过对4个不同钾营养效率的烤烟品种的根皮层细胞质膜内向跨膜钾电流进行全细胞记录,研究钾营养效率不同的烤烟基因型的钾离子通道的特性。结果表明,NC89的电流密度(pA/pF)最高,约为-80;其次是农大202及净叶黄,其电流密度分别为-60及-50;NC2326的电流密度在四个基因型中最小,大约-40。结果表明,烤烟根皮层细胞质膜跨膜内向钾电流的大小,与基因型对K+亲和力及吸收能力的大小,以及烤烟基因型钾营养效率高低有着密切的关系,可以作为一个比较可靠的参考鉴定指标,指导K+营养高效的基因型的筛选与鉴定。     

Acquired temperature-sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila

General locomotor activity decreases with normal aging in animals and could be partially explained by decreases in neuronal function. Voltage-gated Na⁺ channels are essential in initiating and propagating rapid electrical impulses underlying normal locomotor activity and behavior in animals. Isolation of mutations conferring temperature-sensitive (ts) paralysis has been an extremely powerful paradigm for identifying genes involved in neuronal functions, such as membrane excitability and synaptic transmission. For instance, decreased expression of wild-type Na⁺ channels in flies harboring the no-action-potential ( nap ) mutant allele ( mle^napts ) confers rapid and reversible ts paralysis, because of failure of action potential propagation. Here, we report that aging wild-type Drosophila gradually develops an acquired susceptibility to ts paralysis that is indistinguishable from that seen in young ts paralytic mle^napts mutants. Moreover, we show that this general age-dependent susceptibility is also present in mle^napts flies, although the effects are shifted to lower temperature regimes. The mle^napts flies also exhibit decreased lifespan and increased frailty. Paralysis and decreased lifespan of mle^napts flies were partially rescued by increasing the dosage of para , the structural gene for the major action potential Na⁺ channel in central nervous system of Drosophila . Lastly, we show a dramatic scaling of ts paralysis susceptibility with chronological age in short-lived and long-lived mutant flies, further demonstrating that this age-dependent risk is independent of genetic background. Thus, decreased neural transmission, a hallmark of which is ts paralysis, is a biomarker of aging.