Regulation And Mechanism Of K2P Channels Modulators On NLRP3 Inflammasome

BACKGROUNDInflammasomes are the key components of innate immunity.Among a variety of inflammasomes,NLRP3 inflammasome is the most clinically relevant,and its abnormal activation is closely associated with several diseases,including atherosclerosis,Alzheimer’s disease,type 2 diabetes and gout.In the vast majority of cases,the efflux of intracellular potassium ion is a necessary trigger for the activation of NLRP3 inflammasome.However,the mechanisms of potassium ion efflux and NLRP3 inflammome activation remain poorly understood.Two-pore potassium ion(K2P)channels are the last family of potassium channels discovered,mediating the resting membrane potential of cells.In mammals,the K2P channel family comprises of six subfamilies,including TWIK,TASK,TREK,TALK,THIK and TRESK,which are widely distributed in tissues and cells and involved in a variety of physiological or pathological processes.DCPIB(4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl)oxobutyric acid)is a novel inhibitor of TREK channels,which has been found to inhibit NLRP3 inflammasome activation.A recent study has shown that TWIK2 channel-mediated potassium ion efflux is necessary for the activation of NLRP3 inflammasome induced by ATP,and in vivo,deletion of TWIK2 can prevent inflammatory lung injury caused by sepsis in mice.Our study intends to explore new small molecule modulators of K2P channels and the mechanisms,and explor their regulatory effects on NLRP3 inflammasome,so as to provide theoretical basis for drug discovery of NLRP3 inflammasome-associated autoimmune diseases and neurodegenerative diseases.OBJECTIVES1)To explore the regulation of DCPIB on subtype specific K2P channels.2)To explore the molecular mechanism of K2P channels regulated by DCPIB.3)To idetify novel inhibitor of TWIK2 channel and examine its regulatory effect on the activation of NLRP3 inflammasome.METHODS1)By heterologously transfecting K2P channels into COS-7 cells,the regulatory effects of DCPIB on various subtypes of K2P channels were explored.2)By constructing mutants and chimeras and combining with electrophysiological experiments,the molecular mechanisms of K2P channels regulated by DCPIB were preliminarily explored.3)Heterologously transfect TWIK2 channel into COS-7 cells to find novel inhibitor of TWIK2 channel.4)To explore the effect of ML365 on the activation of NLRP3 inflammasome.RESULTS1)DCPIB can activate the currents of TREK 1 and TRAAK channels,meanwhile can inhibit the currents of TRESK,TASK1 and TASK3 channels.2)The effect of DCPIB on TREK1 channel is dependent on the pore domain,while the effect of DCPIB on TRESK channel is independent on the pore domain.3)Through screening six reported modulators of K2P channels,it is found that ML365 can inhibit the current of TWIK2 channel.4)ML365 can inhibit the activation of NLRP3 inflammasome induced by ATP.5)The inhibition of ML365 on NLRP3 inflammsome activation is dependent on TWIK2 channel.CONCLUSIONOur results showed that DCPIB can activate the currents of TREK1 and TRAAK channels.The activating effect is of voltage dependence and the effect of DCPIB on TREK1 is dependent on the pore area.Meanwhile,DCPIB can inhibit the currents of TRESK,TASK1 and TASK3 channels with no voltage dependence.And the effect of DCPIB on TRESK was independent of the pore area.DCPIB has distinct effects on different subtypes of K2P channels,which could become an useful tool for further investigating the structure-function relationship of K2P channels and may serve as a representative novel structure for future design of K2P channel modulators.On the other hand,we found that ML365 can inhibit the current of TWIK2 channel,with an IC50 of 4.069 ± 1.49 μM.In addition,ML365 can specifically inhibit the activation of NLRP3 inflammasome induced by ATP,and the efffect is dependent on TWIK2 channel.ML365 could be a lead compound for future inhibitor of TWIK2 channel,and could provide a theoretical basis for future drug development of NLRP3 inflammasome-associated diseases.