A New Mechanism Of A Novel Antidepressant In Regulating Potassium Channels In Mouse Cortical Neurons

Depression (depression) is a common mood disorder which caused by a variety of reasons. Depressed people may feel sad, anxious, empty, hopeless, helpless, worthless, guilty, irritable, or restless. They may lose interest in activities that once were pleasurable, experience loss of appetite or overeating, or problems concentrating, remembering details or making decisions; and may contemplate or attempt suicide. The study of new antidepressant drugs which started from the1980s developed the second generation of antidepressants. The second generation antidepressant drug efficacy significantly more than the first generation of antidepressant drugs with low anticholinergic effect, little effect on the cardiovascular system and other characteristics, has been described as a high security antidepressants.Recently papers which study the antidepressants are mainly concentrated in reported phenomenon in the efficacy of cardiovascular pharmacology, central nervous system, metabolic regulation and animal behavioral. However, few studies have reported that antidepressants could regulation of the central neuronal membrane ion channels. Moreover, no one knows how antidepressant drugs regulate the cellular and molecular mechanisms of neuronal potassium channels. In our study we use the whole-cell patch clamp recording and brain slice electrophysiological recording, immunohistochemistry, western blot protein quantification tests and molecular cloning techniques and other methods, to explore new antidepressant drug amoxapine, AXP and cyproheptadine (cyproheptadine, CPH) regulate the mouse cortical neurons in the delayed rectifier outward potassium currents by molecular and cellular mechanism. This study shows that the new antidepressant drugs in the regulation of mouse cortical neuronal potassium channel through a new mechanism of indirect regulation by G-protein receptor coupled second messenger to regulate downstream signaling pathways. The study is divided into the following three parts:Part One:We have demonstrated that the antidepressant drug amoxapine suppresses rectifier outward K+(Ik) currents in mouse cortical neurons. At a concentration of10μM to500μM, amoxapine reversibly inhibited Ik currents in a dose-dependent manner and modulated both steady-state activation and inactivation properties. The application of forskolin or dibutyryl cAMP mimicked the inhibitory effect of amoxapine on Ik and abolished further inhibition by amoxapine. H-89, a PKA inhibitor, augmented Ik amplitudes and completely eliminated amoxapine inhibition of Ik currents. Amoxapine was also found to significantly increase intracellular cAMP levels. The effects of amoxapine on Ik currents were abolished by pre-incubation with5-HT. The selective Kv2.1subunit blocker Jingzhao toxin-Ⅲ (JZTX-Ⅲ) reduced Ik amplitudes by30%and also significantly abolished the inhibitory effect of amoxapine. Together these results suggest that amoxapine inhibits Ik currents in mouse cortical neurons by cAMP/PKA-dependent pathways associated with the5-HT receptor, and suggest that the Kv2.1a-subunit may be the target for this inhibition.Part Two:In this part, we used cortical neurons and HEK-293cell transfected with Kv2.1a-subunit to address whether CPH modify neural voltage-gated K+channels by a mechanism unrelated to its serotonergic and histaminergic properties. Our results demonstrate that intracellularly delivered CPH increased the Ik by reducing the intracellular activity of pPKA. The inhibition of Gi eliminated the CPH-induced effect on both the Ik and pPKA. A ligand-receptor binding assay indicated that CPH bound to the sigma-1receptor. Similar results were obtained from HEK-293cells transfected with the a-subunit of Kv2.1. We reveal for the first time that CPH enhances the Ik by reducing the intracellular levels of phosphorylated PKA, and that the associated activation of the sigma-1and the μ-opioid receptor/Gj-protein pathway might be involved.Part three:Finally, we performed visualized current-clamp recordings to determine the effects of CPH on the membrane excitability of mPFC pyramidal neurons in mouse brain slices. Our results demonstrate that intracellularly delivered CPH increased the frequency of action potentail through the sigma-1receptor induced signal pathway. The current-voltage relationship was also altered by CPH showing reduced outward rectification during membrane depolarization and decreased inward rectification during membrane hyperpolarization. Furthermore, CPH enhanced the inward potassium current through the sigma-1receptor induced PKA signal pathway. Mouse open-field behavior test also revealed that CPH could increase the distance of the mouse covered within ten minutes similarly as the cocaine effect. Our findings may help to explain some of the uncharacterized effects of CPH on neuron excitability and contribute to the understanding of CPH as more than just a competitive inhibitor of the histamine H1and serotonin receptors.