The Effects Of Activation Of ATP-Sensitive Potassium Channels On Iron Transport Function Of Dopaminergic Cells And Its Role In Parkinson's Disease

Iron is one of the essential metal elements in all living bodies. It is necessary to maintain iron homeostasis for normal physiological functions. However, excess iron can generate highly reactive hydroxyl radicals (-OH) by the Fenton reaction, which can damage proteins, lipids and DNA. Accumulated evidence showed that increased iron content was observed in the substantia nigra (SN) of Parkinson's disease (PD) patients. PD is a progressive neurodegenerative disorder characterized by selectively dopaminergic neurons loss in the substantia nigra pars compacta (SNpc) and the subsequent dopamine (DA) depletion in the striatum (Str). This disorder is characterized symptomatically by resting tremor, rigidity and bradykinesia. The mechanisms underlying PD pathogenesis have not been revealed yet. Multiple factors might be involved, such as heredity, oxidative stress, environmental factors, inflammation, iron accumulation in SN and cell apoptosis. Due to the toxicity of iron and the ability to produce highly reactive hydroxyl radicals by the Fenton reaction, the selective accumulation of iron in SN plays an important role in the degeneration of dopaminergic neurons.In brain, DA neurons were located in SN, ventral tegmental area (VTA) and arcuate nucleus. But DA neurons in the SN were selective degenerated, but DA neurons in the neighboring VTA are significantly less affected in PD. However, the mechanisms are still unknown. Liss identified selective activation of ATP-sensitive potassium (KATP) channels might be a potential mechanism for this differential vulnerability of DA neurons publicated in Nature Neuroscience in 2005. KATP channels in dopaminergic neurons have been shown to open inducing cell hyperpolarization in response to ATP depletion and increased oxidative stress (ROS) in cells. Our previous studies have showed that elevated iron levels in the SN of PD was induced by the misregulation of iron transporters such as divalent metal transporter 1 (DMT1), indicating DMT1 might be a contributing factor to iron accumulation in PD. It was also reported that iron transport function of DMT1 could be affected by temperature, PH value and hyperpolarization. However, the role of cell hyperpolarization induced by activation of the KATP channels in DMT1 mediated iron accumulation was not clear. In the present study, we investigated the role of KATP channels in the iron transport function of DMT1 and its role in PD using cell culture, whole cell patch clamp recording, molecular biology, immunofluorescence, RT-PCR, western blots and other methods. The main findings are as follows:1. Using DiBAC4(3) to observe the effect of diazoxide (a novel KATP channel opener) on the cell membrane potential, the results showed that the fluorescence intensity was decreased compared with HBS group when perfused with diazoxide in 30 min.2. In the nigral dopaminergic neurons, application of 100μmol/L diazoxide significantly hyperpolarized the membrane potential and decreased the firing rate in tonic firing neurons.3. Using calcein to observe the effect of diazoxide on the iron uptake of SK-N-SH cells, the results showed that the fluorescence intensity was decreased compared with HBS group when perfused with 100μmol/L ferrous iron, indicating the increased iron influx; when perfused with 100μmol/L ferrous iron and diazoxide, the fluorescence intensity was decreased compared with ferrous iron group.4. After pSilencer-DMT1 siRNA expression vector was successfully constructed, we observe the effect of diazoxide on the iron uptake of SK-N-SH cells. Results showed that the fluorescence intensity of pSilencer-DMT1 siRNA group was increased compared with vector group when perfused with 100μmol/L ferrous iron. After perfused with 100μmol/L ferrous iron and diazoxide, the fluorescence intensity of pSilencer-DMT1 siRNA group was increased compared with vector group.5. HEK293 cells were transfected with pcDNA3.1-Kir6.2 vector and pcDNA3.1-SUR1 vector, then infected with AdDMTl+IRE. Whole cell patch clamp recording was used to analyze effects of KCO diazoxide on DMT-1-mediated divalent metal transport. A rapidly activating, inwardly rectifying current was evoked in HEK293 cells when perfusion with ferrous iron, and this current weakened rapidly. In the presence of diazoxide, the current showed a rapidly activation which was stable and weakened slowly. 6. Intracellular iron concentration was measured using an inductively coupled plasma (ICP-2) detector. Results indicated that the intracellular iron concentration was increased in 100μmol/L diazoxide pretreatment group compared with ferrous iron group. To evaluate the intracellularΔΨm and ROS generation, cells were analyzed by flow cytometry. SK-N-SH cells were incubated with 100μmol/L diazoxide for 0.5 h, then the cells were incubated with 100μmol/L ferrous iron and diazoxide for 4 h, results indicated a reduction of the mitochondrial transmembrane potential (ΔΨm) and an increase in reactive oxide species (ROS) generation compared with ferrous iron group.7. SK-N-SH cells were transfected with pcDNA3.1-Kir6.2 vector and pcDNA3.1-SUR1 vector. Over expression of SUR1 and Kir6.2 dramatically increased the ferrous iron influx and iron content in SK-N-SH cells compared to pcDNA3.1 vector alone when treated with 100μmol/L diazoxide and ferrous iron, which induced a decrease inΔΨm and an increase in ROS production.8. SK-N-SH cells transfected with pcDNA3.1-Kir6.2 vector and pcDNA3.1-SUR1 vector or pcDNA3.1 vector were treated with 100μmol/L,200μmol/L and 300μmol/L MPP+for 24 h. In pcDNA3.1 vector cell group, no difference of cell viability was observed when treated with 100μmol/L% 200μmol/L MPP+, a significant reduction of cell viability was observed when treated with 300μmol/L MPP+. In cells over-expressed Kir6.2 and SUR1, there was a significant reduction of cell viability when treated with 100μmol/L MPP+. In addition, no difference ofΔΨm and ROS production were observed when treated with 100μmol/L MPP+in pcDNA3.1 vector cell group. There was a significant reduction ofΔΨm and increase of ROS were observed in cells over-expressed Kir6.2 and SUR1 when treated 100μmol/L MPP+.The selective KATP channels opener diazoxide could hyperpolarize the membrane potential and decreased the firing rate. The hyperpolarization of dopaminergic neurons could increase the iron transport function of DMT 1, and then increase the intracellular iron levels. Due to the toxicity and the ability of free radical production of ferrous iron, elevated intracellular iron levels aggravated cellular oxidative stress. Over expression of SUR1 and Kir6.2 dramatically increased the iron uptake and the oxidative stress damage, strengthened the toxicity induced by MPP+. Our research will provide new theoretical and experimental evidence on for the role of KATP channels in iron accumulation and the selective degeneration in dopaminergic neurons.