Role Of Delayed Rectifier Potassium Channel Kv2.1 Played In Oxygen-glucose Deprivation Induced Cell Insult And The Relevant Pharmacological Study

Cerebral ischaemia is a major cause of disability and death globally and has a profoundly negative impact on the individuals it affects, those that care for them and society as a whole. The most common and familiar manifestation is stroke,85% of which are ischaemic and which is the third leading cause of death after cardiovascular diseases and cancer. Meanwhile, ischemic stroke is the most common cause of complex chronic disability worldwide. Stroke survivors often suffer from long-term neurological disabilities significantly reducing their ability to integrate effectively in society with all the financial and social consequences that this implies.Among clinical trials for ischemic stroke, those involving thrombolytic, anti-thrombotic, anti-platelet agents are by far more numerous than clinical trials of neuroprotectants and these agents protect the brain do so primarily via hemodynamic rather than metabolic mechanisms. Take the ischemic neuron ionic homeostasis imbalance as a cut-in point; Centering on excessive potassium channel activating and intracellular K+ depletion happened in ischemic stroke pathological process, our study investigated the role of slow-inactivating delayed rectifier potassium channel Kv2.1 played in oxygen-glucose deprivation induced cell insult and whether the drug which is already adopted in neurodegenerative diseases clinical trails—donepezil could protect cells against hypoxia/ischemia apoptotic death by blocking Kv2.1 potassium channel independent of acetylcholine receptor system; centering on the function of Na+/Ca2+ exchanger performed in ischemic stroke pathological process, we screened the NCX activators. Our researching is aim at providing the clues and theoretical foundation for the research and development of anti-ischemic stroke neruoprotective agents.Part I:Role of delayed rectifier potassium channel Kv2.1 played in oxygen-glucose deprivation induced cell insult1. Neruoprotective effect of delayed rectifier potassium channel blocker in MCAO ratsIonic homeostasis imbalance leads by energy failure play a critical role in the ischemic neuron damage during cerebral ischemia pathological process. Since excessively loss of intracellular K+is one of the important factor result in ischemia neuron insult, K+channel blockers could attenuated hypoxia/ischemia cell death in cerebral ischemia. In the former research work of our group, we found that rats after middle cerebral artery occlusion and reperfusion, infarction region mRNA expression of Kv2.1 increased dramatically, so we hypothesized that delayed rectifier potassium channel Kv2.1 might be one target for anti-ischemic stroke drugs. Therefore, the effects of delayed rectifier potassium channel blocker TEA on middle cerebral artery occlusion (MCAO) rats were investigated in the present study:compared with vehicle-control group, intracerebroventricular injection TEA (5ug·kg-1) could significantly reduce the infarct volume.2. Kv2.1/HEK293 cells were more susceptible to OGD insult than Wt/HEK293 cellsDelayed rectifier-type potassium channel Kv2.1 is expressed at high levels on all neuronal somata and proximal dendrites of neurons in brain and it has been proved to be a major component of neuron outward delayed rectifier potassium current. Furthermore, Kv2.1 has been suggested as a necessary delayed rectifier K+channel subtype in regulating apoptotic signaling cascade in mammalian cortical neurons in culture. In order to identify the role of Kv2.1 played in oxygen-glucose deprivation induced cell insult and rule out the impact of other channels, we selected the Kv2.1 transfected HEK293 cells. Results showed that the delayed rectifier K+current (IK(DR)) density of Kv2.1/HEK23 cells (416.05±13.45 pA/pF**P<0.01) was more than 10 times higher than that of Wt/HEK293 cells (35.18±4.05 pA/pF). To evaluate the cell resistance of Kv2.1/HEK293 cells and Wt/HEK293 cells to OGD, these cells were exposed to OGD for 2hr,6hr and 12hr. Consistent chemical hypoxia affected the cell viability of Kv2.1/HEK293 cells to a much greater degree than it did in Wt/HEK293 cells. To study OGD induced cell apoptosis, chromatin condensation were evaluated after OGD treatment. Results by Hoechst33342 staining assay showed that hypoxia increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. Compared with HEK293 cells, OGD induced a higher cell apoptosis rate in Kv2.1/HEK293 cells.3. Electrophysiological properties of Kv2.1/HEK293 cells were changed by OGDVoltage-clamp and current-clamp recording mode was applied to detect the current and membrane potential of Kv2.1/HEK293 cells. Results indicated OGD slightly inhibited Kv2.1 current without influencing the kinetic property of this channel, however, OGD exposure could induced a membrane depolarization to around 2mV within a short period of time. At this membrane potential, Kv2.1 could be activated then lead to massive K+efflux and the subsequent cell damage if the membrane potential maintain at this level persistently due to Kv2.1 channel slow inactivation or almost without auto inactivation property after they were activated.4. Effect of K+concentration on isolated mitochondrial membrane potentialMitochondria are intracellular organelles in which high energy phosphate is produced. The role of the mitochondria in apoptotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic death and the stability of mitochondrial membrane potential is important for the decrease of mitochondrial membrane permeability and prevents apoptosis factor release from mitochondrial. Mitochondrial membrane potential (MMP) was measured by mitochondrial uptake of Rhodamin123, a kind of fluorochrome. After the mitochondrial was isolated, they were coincubated in intracellular solution with different K+ concentration. Results showed that:Mitochondrial membrane potential decreased with the reduction of K+concentration in the intracellular solution. This result indicated that the K+concentration is important for the stability of mitochondrial membrane potential.5. Effect of Kv2.1 potassium channel on OGD induced Kv2.1/HEK293 mitochondrial apoptosis factor release.Mitochondria are often centrally involved in the development of apoptosis after cerebral ischemia. Mitochondria are the targets for many intracellular anti-apoptotic and pro-apoptotic signals. In response to pro-apoptotic signals, cytochrome C (Cytc) and apoptosis induce factor (AIF) are released from the intermembrane space. In the cytoplasm, Cytc and AIF could produce further downstream events including the activation of caspase-dependent DNase leading to intemucleosomal fragementation of DNA and ect. Therefore, the release of Cytc, AIF and other mitochondrial proteins from the intermembrane space is commonly a critical step in the death of cells by apoptosis. So in our study, the release of Cytc and AIF into the cytoplasm as a consequence of OGD was detected by Western blotting. Compared with HEK293 cells, OGD could markedly increase the release of Cytc from mitochondria after 6h treatment in Kv2.1/HEK293 cells and similar result was observed in AIF release. These results confirmed the effect of Kv2.1 on OGD induced mitochondrial apoptosis and suggested that the antiapoptotic ability of Kv2.1 blocker might be performed partially by suppressing Cytc and AIF release after OGD treatment.Part II:Donepezil attenuate oxygen-glucose deprivation insult through blocking Kv2.1 potassium channel on transfected HEK293 cellsDonepezil is a widely used drug that improves cognitive and global functions in patients with mild, moderate Alzheimer's disease, as well as vascular dementia. Besides its acetylcholinesterase inhibition ability, neuroprotective effect of donepezil has also been supported by the results of many preclinical studies in hypoxia/ischemia insult models, such as middle cerebral artery occlusion (MCAO) in rats; oxygen-glucose deprivation, glutamate, and Nmethyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)/kainite-induced excitatory amino acids insult, veratridine induce membrane depolarization insult using primary-cultured neurons. These results imply that the neuroprotective effect of donepezil may not be totally dependent on acetylcholinesterase inhibitory activity and the precise neuroprotection mechanism of donepezil is remaining to be clarified. However, interference with the K+-dependent cell damage pathway may be one of them; because among all the above injury models, neurons suffer from sustained membrane depolarization and subsequent excessive K+efflux.1. Donepezil inhibited Kv2.1 currents in a dose-dependent mannerThe Kv2.1 current did not alter significantly during the recording in the time matched control (without donepezil), so the run-down of current could be ruled out. In the presence of 30μM of donepezil, Kv2.1 currents were inhibited to 49.93±6.59%(n=10) of control and the current density of Kv2.1 decreased from 412.42±37.76 pA/pF to 179.63±26.95 pA/pF. Analysis of the concentration-response relationship revealed an IC50 value of 7.59μM. Meanwhile, lOmM TEA decreased Kv2.1 current density from 360.05±53.81 pA/pF to 153.76±25.23 pA/pF.2. Effect of donepezil on OGD Kv2.1/HEK293 cell Electrophysiological propertyOGD induced membrane depolarization is sure to lead to activation of Kv2.1 potassium channel. OGD could decreased Kv2.1 currents amplitude, but this blockade effect of OGD on Kv2.1 currents was not as potent as donepezil did. In the presence of 30μM of donepezil, the current density of OGD Kv2.1 decreased from 354.80±19.25 pA/pF to 123.79±22.69 pA/pF while the depolarizing test pulse was to+60mV.Membrane potential of Kv2.1/HEK293 cells were moved toward to depolarization direction after OGD treatment. In the presence of 30μM of donepezil and lOmM TEA, OGD induced membrane depolarization was decreased for 5-7mV. Although donepezil and TEA attenuated the membrane depolarization induced by OGD, membrane potential at around 0mV still could result in the activation of Kv2.1 potassium channel.The steady-state activation curve of Kv2.1 showed that donepezil didn't shift the OGD Kv2.1 half-maximal activation potential (V1/2) significantly. Whereas, donepezil at 30μM significantly shifted the inactivation curve to negative potential by 8.73mV, these results implicated that donepezil blocked Kv2.1 channel mainly due to accelerating the inactivation of this channel and further inhibited the massive K+loss induced by OGD.3. Donepezil attenuate OGD induced apoptotic damage in Kv2.1/HEK293 cellsMTT staining and Hoechst33342 dye were used to assess the cell viability and apoptois. HEK293 cells become more susceptible to OGD insult after they were transfected with Kv2.1 potassium channel. Donepezil 30μM significantly increase the viability of OGD insulted Kv2.1/HEK293 cells. Since the blockade effects of 30μM donepezil on normal and OGD insulted kv2.1 currents is close to that of 10mM TEA, we observed the antiapoptotic effect of donepezil with TEA at this concentration. In the present study, donepezil and TEA significantly attenuated OGD induced apoptotic morphological alteration, including unhealthy bodies and chromatin fragmentation.4. Donepezi inhibit OGD induced mitochondrial apoptosis factor release in Kv2.1/HEK293 cellsOGD markedly increased the release of Cytc and AIF from mitochondria into cytoplasm after 6h treatment in Kv2.1/HEK293 cells. In present of 30μM of donepezil, OGD induced the release of Cytc and AIF from mitochondria decreased significantly. These results imply that the anti-OGD-induced apoptotic effect of donepezil maybe contributed to its ability that blocked Kv2.1 potassium channel then stabilizing mitochondria membrane potential, inhibited Cytoc and AIF form mitochondria; and thereby inhibited cell apoptosis, promoted the survival of Kv2.1/HEK293 cells in response to OGD damage.5. Effect of donepezil on OGD Kv2.1/HEK293 cell cytoplasm PKC-delta levelPKC-delta is a member of the novel PKC family that can be activated in response to numerous cellular stimuli by various mechanisms. The activation pathways include membrane translocation, tyrosine phosphorylation or proteolytic cleavage by an activated enzyme and activated PKC-delta participate in cell apoptosis procedure. By detecting the decrease of complete from PKC-delta, we can observe PKC-delta activation extent. In our research, Kv2.1/HEK293 cell complete from PKC-delta level in cytoplasm decreased signifcantly after 6hr of OGD treatment; but both donepezil and TEA didn't impact the decreased complete from PKC-delta level in cytoplasm. This result imply that anti-OGD-induced apoptotic effect of donepezil may not correlate with PKC-delta activation pathway.PartⅢ:Anti-ischemic stroke drug screen based on NCX functionThe Na+/Ca2+exchanger (NCX) is a bi-directional membrane ion transporter. Under normal physiological conditions, the exchanger transports one calcium ion out of the cell and three sodium ions into the cell. Because dysregulation of sodium and calcium homeostasis is an integral feature of ischemic brain injury, the role of the NCX in neurons following ischemia has been investigated. Studies using in vitro ischemia-related models have produced conflicting results. However, the majority of in vivo studies using the focal cerebral ischemia model and NCX transgenic animals indicate that blocking NCX activity or knocking out NCX gene is neurodamaging while increasing NCX activity is neuroprotective, especially targeting at NCX3. In our research, we studied 9 compounds on NCX1, NCX2 and NCX3 in transgenic cells using Port-a-patch whole cell patch-clamp techniques. Results:4 compounds were found to be the activator of all three NCX isoform,6 compounds could active NCX3 and further study is needed on these compounds in ischemic stoke models to explore promising anti-cerebral ischemia drugs.