I. Introduction 1 II. MATERIALS AND METHODS 9 Cell culture and transfection 9 Dissociation of superior cervical ganglia 9 Electrophysiological recording 10 Confocal imaging and analysis 11 Förster Resonance Energy Transfer (FRET) 11 Solution and materials 12 Non-stationary noise analysis 12 Animals and tissue collection 13 Dissection and dissociation of primary hippocampal neurons 14 Western blot analysis 14 RNA extraction and real-time PCR assays for miRNA and mRNA 15 Plasmid construction 16 Image acquisition 16 Data analysis and statistical analysis 17 III. RESULTS 18 3.1. Ethanol Elevates Excitability of Superior Cervical Ganglion Neurons by Inhibiting Kv7 Channels in a Cell Type-Specific and PI(4,5)P2-Dependent Manner 18 SCG neurons can be classified into phasic-1, phasic-2, and tonic neurons based on their action potential firing patterns and IKv7 size 18 Excitability and the resting membrane potential of SCG neurons are affected by inhibiting Kv7 channels in a cell type-specific manner 21 Ethanol increases the excitability of SCG neurons 23 Ethanol inhibits Kv7 channels in cultured SCG neurons as well as the heterologous Kv7.2/7.3 channels expressed in tsA201 cells 26 The inhibition of IKv7 by ethanol is not due to either degradation or translocation of plasma membrane PI(4,5)P2 29 Inhibitory effect of ethanol on IKv7.2/7.3 is modulated by the level of the plasma membrane PI(4,5)P2 31 Ethanol inhibition of the Kv7.2/7.3 current is the result of reduction in PI(4,5)P2 binding affinity of the Kv7.2/7.3 channel 36 Ethanol inhibition of Kv7.2/7.3 channels differs from block with internal TEA 40 Ethanol reduces the open probability of the Kv7.2/7.3 channel 44 3.2. Post-transcriptional control of KCNQ2 gene expression in brain by miRNAs 47 Kv7.2 channel expression is up-regulated by miR-106b family during early developmental stage in mouse hippocampus 47 M-currents are inhibited by up-expression of miR-106b-5p and enhanced by its downregulation in hippocampal neurons 50 miR-106b-5p regulates hippocampal neuronal excitability 54 IV. DISCUSSION 63 V. REFERENCES 72 VI. 국문요약 81
Research Interests
Molecular mechanisms of epilepsy and sensory pain transmission; Signaling mechanism of ion channel regulation and membrane excitability; 분자전기생리; 간질 및 통증의 분자적 기전 연구