Screening TREK1 Channel Blocker And Exploration Of Its Antidepressant Mechanism

Background and Objective:One of the prominent problems with antidepressant treatment is that current antidepressants often remain the inadequate efficacy for many depressive patients. Recently, the two-pore domain potassium channel TREK1 has been confirmed as an antidepressant target related to therapeutic effect. In addition, the star*D study has demonstrated an association between genetic variation in KCNK2 and resistance to antidepressant treatment. However, desirable TREK1 blocker has been not discovered, and antidepressant mechanism of TREK 1-mediated response is not clear. This study, based on screening a specific TREK1 blocker, observes the antidepressant-like effect of screened TREK1 channel blocker, designs to explore the antidepressant-like mechanism.Methods:1. TREK1-pCDNA3.1(+)/pEGFP-N1 plasmids were constructed by molecular clone technology and transfected in human embryonic kidney (HEK293) cells to express TREK1 in vitro.2. TREK1 blocker was screened by thallium mediated high throughout screening (HTS) from a chemical library of Shanghai Institute of Materia Medica affiliated Chinese Academy Sciences, and screened TREK1 blocker was evaluated the sensitivity and specificity by whole cell patch-clamp.3. The interaction of SED1900 and TREK1/5-HT1A receptor was measured using surface Plasmon Resonance (SPR).4. Effective dose of screened TREK1 blocker was calculated by pharmacokinetic experiment.5. Screened TREK1 blocker, spadin and citalopram were evaluated the antidepressant response on chronic unpredictable mild stress model by force swim test (FST), open field test (OFT) and sucrose preference test (SPT).6. Effects of blocking TREK1 on 5-HT ergic and pyramidal neuronal field potential were observed in dorsal raphe nucleus (DRN), prefrontal cortex (PFC) and hippocampus CA1, CA3 of depressive model by electrophysiological experiment.7. Expression of protein kinase A (PKA), cAMP response element binding protein (CREB) and brain derived neurotrophic factor (BDNF) were examined in DRN, PFC and hippocampus CA1, CA3 by wastern blotting and real-time PCR.8. Effects of 5-HT1A receptor (5-HTR1A) agonist 8-OH-DPAT and antagonist WAY 100635 on TREK1 current were observed by whole cell patch-clamp, meanwhile 8-OH-DPAT and WAY 100635 were used to incubate primary-cultured rat hippocampal neurons at the present of screened TREK1 blocker or reported TREK1 blocker to examine expression of PKA, CREB and BDNF by Western blotting.Results:1. The IC50 value for inhibition of the TREK1 channel fluorescence by SID 1900 was 28.72±3.67μM in HTS. Electrophysiological data demonstrated that SID 1900 blocked efficiently TREK1 current in TREK1-pEGFP-N1-transfected HEK293 cells and IC50 value was of 29.72±2.4μM at OmV. It is crucial that SID 1900 blocked selectively two-pore domain potassium channel (p<0.001, IC50=87.09±4.2μM) instead of Na+, mix K+ and Ca2+ channel currents in hippocampal neurons (p>0.05).2. SPR kinetic tests with increasing concentrations of SID1900 showed that SID1900 had high affinity to TREK1, with balance dissociation constant (KD) 1.905×10 -10 M.3. Pharmacokinetic data revealed SID1900 with satisfactory blood brain barrier (BBB) permeation rate (78.74%) and biological availability (78.03±9.65%), and effective dose of SID 1900 was 14.33 μM/kg in animal experiment.4. Two weeks-treated with SID 1900 rats showed a resistance to depression in FST, SPT and OFT the same as spadin, compared with that undergoing treatment with citalopram (p<0.05). 5. The electrophysiological experiments in vivo indicated that SID 1900 enhanced obviously firing rates of 5-HT ergic neuron in DRN (4.35±0.61) Hz and pyramidal neuron in PFC (5.02±0.82)Hz, compared with CUMS rats (p=0.014, p=0.003) and citalopram (p<0.05), which accorded with spadin. In addition, acute administration of SID 1900, spadin and citalopram increased significantly the firing rate of DRN 5-HT neurons at 6 min,7min40s and 9min40s in the CUMS rats respectively.6. The westernblotting and real-time PCR data manifested that a 2 weeks treatment with SID1900 upregulated obviously expression of PKA-CREB-BDNF signaling in DRN, hippocampus CA1/CA3 and PFC of CUMS-rats compared with CUMS-depressive rats (p<0.001), and the similar effect were observed in 2 weeks-treated rats with spadin and 4 weeks-treated rats with citalopram.7.30μM 5-HTR1A agonist 8-OH-DPAT amplified 19.14±1.34%(n=6) of TREK1 currents measured at 0 mV, and 10μM 5-HTR1A antagonist WAY 100635 blocked 17.18±1.26% of TREK1 currents measured at 0 mV. In addition, a 1day treatment with SID 1900 and 8-OH-DPAT upregulated obviously expression of PKA-CREB-BDNF signaling in primary-cultured hippocampal neurons compared with single SID 1900 treatment (p<0.05), meanwhile a 1day treatment with SID 1900 and WAY 100635 could down-regulated significantly expression of PKA-CREB-BDNF (p<0.05). In addition, SPR kinetic tests with increasing concentrations of SID 1900 showed that SID 1900 had low affinity to 5-HT1A receptor, with balance dissociation constant (KD)2.597×10-4M.Conclusion:1. Screened SID1900 might block efficiently TREK1, and has specificity to K2P channel in neuron.2. SID 1900 with satisfactory BBB permeation rate and biological availability induces a significant antidepressant-like response.3. SID1900 blocks TREK1 to upregulate PKA-CREB-BDNF signaling in neuron synergizing with 5-HT1A receptor.4. Antidepressant-like action of SID 1900 could be related with increasing the transmission of 5-HT, synergic role of 5-HTR1A and up-regulation of PKA-CREB-BDNF expression in DRN, hippocampus CA1/CA3 and PFC.