ABSTRACT
TWIK (tandem-pore domain weak inward rectifying K(+))-related spinal cord K(+) channel, TRESK, a member of the tandem-pore domain K(+) channel family, is the most recently cloned K(2P) channel. TRESK is highly expressed in dorsal root ganglion neuron, a pain sensing neuron, which is a target for analgesics. In this study, a reliable 3D structure for transmembrane (TM) region of mouse TRESK (mTRESK) was constructed, and then the reasonable blocker binding mode of the protein was investigated. The 3D structure of the mTRESK built by homology modeling method was validated with recommend value of stereochemical quality. Based on the validated structure, K(+) channel blocker-bound conformation was obtained by molecular docking and 5ns MD simulation with DPPC lipid bilayer. Our docking study provides the plausible binding mode of known blockers with key interacting residues, especially, F156 and F364. Finally, these modeling results were verified by experimental study with mutation from phenylalanine to alanine (F156A, F364A and F156A/F364A) at the TM2 and TM4. This is the first modeling study for TRESK that can provide structural information of the protein including ligand binding information. These results can be useful in structure based drug design for finding new blockers of the TRESK as potential therapeutic target of pain treatment.
Subject(s)
Potassium Channels/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Alanine/chemistry , Amino Acid Sequence , Animals , Binding Sites , DNA Mutational Analysis , Electrophysiology/methods , HEK293 Cells , Humans , Ions , Ligands , Lipid Bilayers/chemistry , Mice , Molecular Conformation , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenylalanine/chemistry , Protein Conformation , Sequence Homology, Amino Acid , TransfectionABSTRACT
Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50µM) for different durations. When late 2-cells were incubated with 5µM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5µM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5µM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating.
Subject(s)
Embryonic Development/drug effects , Fluoxetine/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , Animals , Blastocyst/drug effects , Blotting, Western , Calcium Signaling/drug effects , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Chromosomes/drug effects , Enzyme Activation/drug effects , Female , Membrane Potentials/drug effects , Mice , Oxygen Consumption/drug effects , Patch-Clamp Techniques , Polymerase Chain Reaction , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Pregnancy , RNA, Small Interfering/genetics , Signal Transduction/drug effectsABSTRACT
Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.
Subject(s)
Blastocyst/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Potassium/metabolism , Zygote/metabolism , Animals , Blastocyst/drug effects , Embryo Culture Techniques , Embryonic Development , Gene Expression Regulation, Developmental , Hydrogen-Ion Concentration , Immunohistochemistry , Ion Channel Gating , Membrane Potentials , Mice , Mice, Inbred ICR , Potassium Channel Blockers/pharmacology , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Potassium Channels, Tandem Pore Domain/genetics , RNA Interference , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Zygote/drug effectsABSTRACT
1. Two-pore domain K⺠(K(2P) ) channel expression influences brain development. The K(2P) channels, including two-pore domain acid-sensitive K⺠(TASK) channels, contribute to the setting of the resting membrane potential of neurons. In addition to neurons in the brain, dorsal root ganglion (DRG) neurons also express K(2P) channels. The aim of the present study was to identify postnatal changes in the expression of TASK channels in DRG neurons. 2. Expression of TASK channels (TASK-1, TASK-2 and TASK-3) was compared between neonatal (postnatal Day (P) 1 or P2) and adult (P120) rat DRG using semiquantitative polymerase chain reaction, western blot analysis, immunostaining and the patch-clamp technique. 3. In adult (P120) rat DRG, expression of TASK-2 mRNA and protein was downregulated, whereas TASK-3 mRNA and protein expression was upregulated. There were no consistent changes in TASK-1 mRNA and protein expression. Single-channel recordings showed very low TASK-2- and TASK-3-like channel expression in P1-P2 DRG neurons (â¼10% in TASK-2 and â¼3% in TASK-3). In P120 DRG, there was a reduction in the detection of TASK-2-like channels, whereas the detection of TASK-3-like channels increased. 4. These results show that TASK-2 and TASK-3 mRNA and protein expression undergoes age-related changes in DRG neurons, indicating that TASK-2 and TASK-3 channels are likely to contribute to the setting of the resting membrane potential of DRG neurons in neonates and adults, separately or together, during DRG development.
