Inhibitory effect of antidepressant metergoline on Kv1.4 channel 1, 1 Sanung Eom , Khoa Nguyen , Jaeeun Lee , Chaelin Kim , Shinhui Lee and Jun-Ho Lee * 1 1 1 1 1 Department of Biotechnology, Chonnam National University, Gwangju, Korea ABSTRACT RESULTS Figure 1. Effects of metergoline on wild-type Kv1.4 and Figure 5. Effect of metergoline on mutant Kv1.4 D2-61 Voltage-gated potassium channels (VGKCs) are transmembrane ion Kv1.4 D2-61 channel currents. channel. channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a
representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N- terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 D2-61) and found that it induced an outward peak and steady-state currents.
Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 D2-61 channels with acceleration current mode. The Figure 2. Concentration response curves and acceleration of steady-state current of deleted Kv1.4 mutant channel inactivation kinetics of metergoline-induced inhibitory Figure 6. Effects of metergoline on Kv1.4 D2-61 channel in occurred in a concentration-dependent manner. This means that effects on Kv1.4 D2-61 channel currents.
different concentration of extracellular [K+]. metergoline can accelerate C pattern inactivation of Kv1.4 D2-61 channel by acting as an open state dependent channel blocker. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand. MATERIALS AND METHODS Cell preparation Xenopus laevis (Xenopus I, Ann Arbor, MI, USA) Figure 7. Effects of metergoline on Kv1.5 channel and A type K+ current of rat neuronal cell. In vitro transcription and expression In vitro transcription kit
(mMessage mMachine; Ambion) cDNA encoding human genes were transcribed cRNA as protocols cRNA (10-40ng/40nl), Automatic Oocyte injector (Drummond Scientific), Incubation (18℃, 2-3day) Figure 3. Computational molecular modeling of metergoline docked to Kv1.4 channel. Recording and Data analysis Two-microelectrode voltage-clamp recording (Oocyte Clamp (OC- 725C), Digidata 1200A) Figure 8. Comparison of metergoline and quinidine for Kv1.4 D2-61 channel activity regulation. Molecular docking studies Autodock Tools (version 1.5.6) by The Scripps Research Institute (La Jolla, CA) Odorant receptor of
Apocrypta bakeri, Protein Data Bank (ID code B0FAQ4, 3.5A resolution) 3D structure of the ligand (Compound A) was obtained from Pubchem Figure 4. The binding pocket and docking results of metergoline and Kv1.4 channel. SUMMARY AND CONCLUSION In this study, we found that metergoline inhibited K+ channel in voltage- and time-dependent manners, leading to dynamic structural change of channel protein. The current study examined the relationship between metergoline binding and C-type inactivation and showed that metergoline influenced C-type inactivation in voltage gated K+ channel. In conclusion,
metergoline can inhibit Kv1.4 D2-61 channels in a concentration-dependent manner. NT deletion experiments were performed to further characterize effects of metergoline on Kv1.4 channel current. Our results suggest that metergoline can regulate non-inactivating Kv1.4 D2-61 channel currents and increase C-type inactivation rate. These novel findings demonstrate the pharmacological effect of metergoline at cellular and molecular levels. [Q. Neuroscience-1] Inhibitory effect of antidepressant metergoline on Kv1.4 channel Jun-Ho Lee¹ ¹Biotechonology, Chonnam National University, Gwangju 60086,
Korea Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern
inactivation. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human
Kv1.4 channel, providing a novel interaction ligand. Characterization of dysregulated subcellular localization of disease- related proteins of neurodegenerative diseases in Drosophila Yeonjin Jeong, Jeong Hyang Park, Chang Geon Chung, Sung Bae Lee Brain and Cognitive Sciences, DGIST (Daegu Gyeongbuk Institute of Science & Technology), Daegu 42988, South Korea BACKGROUND & AIM Proteins serve as a biological functional unit within the cell, which carefully regulates both the function and translocation of various proteins to maintain protein homeostasis. The translocation of cytoplasmic proteins
to the nucleus has key functional importance within the neuron as the translocation plays a fundamental role in gene regulation and other biological functions. Accordingly, control of protein nucleocytoplasmic transport (NCT) is required for gene regulation. In addition, proteins and interacting partners have different functions and efficiencies depending on protein localization. Therefore, the protein subcellular localization provides a physiological context for their function. According to these reasons, fine regulation of protein subcellular localization regulated by NCT is important. More
importantly, the subcellular mislocalization of protein is easily found in human diseases. It has been reported that several aberrations caused mutation, altered expression of cargo proteins and transport receptors or by deregulation of components of the trafficking machinery contribute to the mislocalization of important proteins. Of note, recent evidence indicates that the aberrant translocation of proteins is seen in most neurodegenerative diseases. The representative examples are follows: TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis (ALS),
tau in Alzheimer’s diseases (AD), α-synuclein (α-Syn) in Parkinson’s disease (PD), huntingtin (Htt) protein in Huntington’s disease (HD), ataxin (ATXN) protein in Spinocerebellar ataxia(SCA). Among these, TDP-43 is one of the most studied neurodegenerative disease. Because TDP-43 show cytoplasmic mislocalization in more than 97% of ALS patients, the mislocalization of TDP-43 is commonly referred to as an ALS disease hallmark. This observation has garnered great attention and research to understand the NCT machinery involved with TDP-43 transportations. To figure out additional disease-related
proteins, other than TDP-43, of which the translocation is regulated by calcium in neurons, candidate proteins were listed. RESULTS Table 1. Disease-related protein candidates for identification of Figure1. Overall scheme for selection of potential disease- which translocation is regulated by Ca 2+ in neurons related proteins that has mislocalization in disease condition Figure2. Subcellular localization of neurodegenerative disease-related proteins in C4 da neurons. (A) Subcellular localization of ATXN3, dFUS, dhnRNPA1, dhnRNPA2, and RpL proteins (red). (B) Quantification of the fluorescent
intensity of proteins(red) in C4 da neurons region illustrated in (A). N.S., not significant; ****P<1.0 x 10 by unpaired t test; error bars, SEM; n ≤ 6 neurons. -4 Contact information To whom correspondence should be addressed. Email: sblee@dgist.ac.kr [Q. Neuroscience-3] Characterization of dysregulated subcellular localization of disease-related proteins of neurodegenerative diseases in Drosophila Yeonjin Jeong¹, Jeong Hyang Park¹, Chang Geun Chung¹, Sung Bae Lee¹˙* ¹Brain and Cognitive Sciences, DGIST (Daegu Gyeongbuk Institute of Science and Technology), Daegu 42988, Korea For proper
functioning of proteins, subcellular localization of proteins is very important. In fact, dysregulation of subcellular localization appears to be associated with many diseases. Particularly, mislocalization of disease-related proteins, such as TDP-43 and polyQ proteins, has been well recognized in neurodegenerative diseases. However, our understanding on what cellular condition facillitates this mislocalization and what are underlying cellular and molecular mechanisms remains largely elusive. In this study, we observed dynamic changes in subcellular localization of disease-related proteins for
neurodegenerative diseases along development in Drosophila neurons and characterized underlying cellular mechanisms. We believe that our findings will be of help for better understanding of pathophysiology underlying neurodegenerative diseases associated with protein mislocalization. MiR-30 and miR-153 alleviate LPS induced inflammation by targeting NeuroD1 in microglial cells. 2* 1 1 Hye-Rim Choi , Ji Sun Ha , Sung-Woo Cho , Seung-Ju Yang 1* 1 Department of Biomedical Laboratory Science, Konyang University, Daejeon, 35365, Korea 2 Department of Biochemistry and Molecular Biology, University
of Ulsan College of Medicine, Seoul, Korea BACKGROUND AIM Neurogenic differentiation 1 (NeuroD1) is a basic helix-loop-helix (bHLH) In the present study, we aimed to analyze the immunoregulatory transcription factor that plays an important role during neuronal differentiation, mechanisms of NeuroD1 in BV-2 cells induced by LPS. Therefore, we maturation and survival. It is reported that NeuroD1 is associated with confirmed anti-inflammatory effects by inhibiting NeuroD1, which is inflammatory response in LPS-induced BV-2 cells. MicroRNA (miRNA) is increased in LPS-induced BV-2 cells. Moreover,
NeuroD1 targeting endogenous small non-coding RNAs consisting of 22 nucleotides that miRNAs are constructed and assessed its effectiveness. After then, we effectively regulates gene expression at the translation level. It is related to a investigate the anti-inflammatory effects of miRNAs against LPS induced variety of central nervous system diseases and microglia differentiation. inflammation responses and NLRP3 inflammasome activation in BV-2 cells. METHODS 1) Cell culture : BV-2 cells were maintained in DMEM with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% FBS in a humidified
incubator at 37°C in 5% CO 2 . 2) Plasmids and transfection : Cells were transfected with NeuroD1 shRNA, scramble shRNA and microRNA using lentiviral packaging vector. Cells were incubated for 24 h before other experiments. 3) Western blotting : BV-2 cells were lysed using RIPA buffer. Proteins were separated on 10% - 12% SDS gels by electrophoresis. The proteins were transferred onto a nitrocellulose membrane, and then incubated overnight at 4℃ with the primary antibody, followed by incubation for 1h at room temperature with secondary antibody. 4) Real-time PCR : RNA were extracted using
Trizol reagent according to manu
