| Summary: | 博士 === 國立中興大學 === 生命科學系所 === 101 === In nervous system, the neurons and astrocytes are very important cells with a lot of physiological functions and expression of P2X7R and voltage-gated K+ (Kv) channels. Overexpression or activation of P2X7R or Kv channels caused cell apoptosis and neuronal diseases, for example, stroke, cerebral ischemia and Alzheimer''s disease. P2X7R activation by extracellular ATP triggers influx of Na+ and Ca2+, cytosolic Ca2+ overload and consequently cytotoxicity. Whether disturbances in endoplasmic reticulum (ER) Ca2+ homeostasis and ER stress are involved in P2X7R-mediated cell death is still unknown. In this thesis, a P2X7R agonist (BzATP) was used to activate P2X7R in differentiated NG108-15 neuronal cells. In a concentration-dependent manner, application of BzATP (10-100 μM) immediately raised cytosolic Ca2+ concentration ([Ca2+]i) and caused cell death after a 24-h incubation. P2X7R activation for 2 hrs did not cause cell death but resulted in a sustained reduction in ER Ca2+ pool size. Furthermore, P2X7R activation (2 h) led to the appearance of markers of ER stress p-eIF2α and CHOP and apoptosis (cleaved caspase 3). XeC, an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor (IP3R), strongly inhibited BzATP-triggered [Ca2+]i elevation, suggesting that the latter involved Ca2+ release via IP3R. XeC pretreatment not only attenuated the reduction in Ca2+ pool size in BzATP-treated cells, but also rescued cell death and prevented BzATP-induced appearance of ER stress and apoptotic markers. Further, Kv channels serve to repolarize during action potentials in excitable cells. Astrocytes are considered non-excitable cells since they are not able to generate action potentials. Intriguingly, astrocytes possess abundant Kv channels; the latter’s functions in astrocytes are hitherto unknown. In this thesis, it was found that astrocytes have both A-type and delayed rectifier type Kv channels. Blockade (20 mM TEA and 30 μM quinidine) of both Kv channels dramatically increased current injection-triggered membrane potential overshoot, curbed ionomycin-induced and store-operated Ca2+ influx. Blockade of Kv channels suppressed proliferation in astrocytes. Further, interaction between the selectivity filter and the adjacent pore helix of Kv channels controls pore stability during K+ conduction. Kv channels, having their selectivity filter destabilized during depolarization, are said to undergo C-type inactivation. The functionality of a residue at the pore helix of the Kv1.2 channel (V370), which reportedly affects C-type inactivation, was examined. A mutation into glycine (V370G) caused a shift in reversal potential from around -72 to -9 mV. The permeability ratios (PNa/PK) of the wild type and V370G mutant are 0.04 and 0.76, respectively. In the wild-type, PRb/PK, PCs/PK and PLi/PK are 0.78, 0.10 and 0.05, respectively. Kv1.2 V370G channels had enhanced permeability to Rb+ and Cs+ (PRb/PK and PCs/PK are 1.63 and 1.18, respectively). Therefore, these novel observations suggest that P2X7R activation not only made Ca2+ overloaded but also rescued cell death and prevented BzATP-induced appearance of ER stress and apoptotic markers. Kv channels regulate Ca2+ signals, membrane potential and proliferation in astrocytes. In addition to its known effect on pore stability, V370 of Kv1.2 is also crucial in controlling ion selectivity. These results indicate that regulation of P2X7R and Kv channels could serve as therapeutic target in neuronal disease in the future.
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