The Research Of K_2P Channel Gating Mechanism And Discovery Of Its Small Molecules

Two-pore-domain K⁺(K_2P)channel is a kind of ion channel encoded by the KCNK gene family which are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem.In the resting state,K_2Pchannels give rise to background or leak potassium currents which maintain the membrane potential and control the cell excitability.K_2Pchannels are widely expressed in the nervous system and are involved in a variety of physiological and pathological processes such as pain,depression,neuroprotection,epilepsy,and ischemia.At present,there are extensive researches on the gating mechanisms of K_2Pchannels including C-type gate.K_2Pchannel is affected by the regulation of intracellular domains.K_2Pchannel is also regulated by external stimulus such as voltage,mechanism force and temperature.However,the specific gating mechanism of K_2Pchannels is still unclear,including the linkage mechanism between the internal structural domains of the channels and selectivity filter.Meanwhile,the paucity of small molecule modulators of K_2Pchannels limits the research on the functional target of K_2Pchannels.Molecular dynamics(MD)simulation and patch clamp electrophysiology are powerful approaches to study ion channels.MD simulation can research the dynamic conformational changes of protein at the atomic level,which provides important clues for the functional studies of protein.Patch clamp technology is the“gold standard”for studying the mechanism of small molecule discovery and regulation of ion channels by detecting and recording the electrical activity of ion channels.The current thesis mainly used the above experimental methods to systematically study the gating mechanism of TREK-1 channel in K_2Pfamily,the mechanism of single channel detection of active compound in the K_2Pchannel,the exploration of structure-activity relationship of antidepressant active inhibitors of TREK-1 and the molecular screening and discovery of novel inhibitors of K_2Pchannel.In order to study the linkage mechanism between the motion of intracellular transmembrane domain in the TREK-1 channel of K_2Pchannel family and the selectivity filter,we used MD simulation to research that the motion of the transmembrane helical region of TREK-1 channel can affect the opening and closing of selectivity filter and then regulate the C-type gating of channel.Cysteine crosslinking and electrophysiological tests also confirmed that the movement of TREK-1 channel transmembrane can regulate C-type gating in the SF region of the channel.We studied the mechanism of agonist CHET3 on TASK-3 channel and agonist C3001a on TREK-1 channel and confirmed the binding site of these two exogenous small molecules on K_2Pchannel,which provide a research basis for following research on the mechanism of action of small molecules on channel.TREK-1 channel has become an important ion channel target for antidepressant effect.It's significant to find specific inhibitors of TREK-1 channel for the treatment of depression and other diseases.Based on structure-based drug design(SBDD),we designed and modified the anti-depression active molecule TKDC.And then we screened and tested the inhibitory effect of TKDC derivatives on TREK-1 channel.Finally we obtained derivative 5f,6f and 6g with better inhibitory activity.Through the structure-activity relationship we found that the 4-chlorophenyl group of derivative 5f can form hydrophobic interaction with residues Q76 and I80 located in the extracellular cap domain of TREK-1.The bicyclic group of 5f also form hydrophobic interaction with Q83,F87,L102 and Q105.It make 5f exert a better inhibitory effect on the binding site of extracellular region of TREK-1,thus blocking the extracellular potassium ion conduction.Through the screening and discovery of novel small molecule inhibitors targeting TASK-3 channel,an inhibitor FJ-03 of TASK-3 channel was obtained.We verified the binding site of FJ-03 in TASK-3 channel.And it was found that FJ-03 was bound to the lower part of the selectivity filter of the channel.A series of TREK-1 inhibitors were found in the screening of novel small molecular for TREK-1 channel.Among them,the inhibit effect of compound TB1Y19 was relatively high.These novel antagonists of the TASK-3 and TREK-1 channels can represent lead compounds and lead to a solid foundation for development of safe and effective clinical antidepressants.