Modulation Mechanism And Novel Inhibitors Exploration Of HCN Channels

BACKGROUNDHyperpolarization-activated cyclic nucleotide-gated channels(HCN channels)belong to the superfamily of voltage-gated ion channels and have unique electro-physiological functions.In mammals,there are four different isoforms of HCN chan-nels,HCN1-HCN4,which are widely distributed in the cardiovascular and nervous system and involved in different physiological processes.It is important to elucidate the mechanism of modulating HCN channels by other signaling pathways and dis-cover compounds targeting HCN channels.Phosphatidylinositol 4,5-bisphosphate(PIP2)is one of the most important mem-brane phospholipids,which can be regulated by multiple receptors and intracellular signaling channels.Recent researches have shown that PIP2 regulates the function of multiple types of ion channels and one of the research contents of this topic is to ex-plore whether PIP2 and its downstream signaling pathways could regulate the func-tion of HCN channels.In addition,HCN channels play a key role in the control of pacemaker activity in the heart and nervous system and hence HCN channels have become a new target for the development and treatment of angina pectoris,pain and epilepsy as drug intervention.Moreover,another research contents of this topic is to screen a series of candidate compounds,aiming to find inhibitors of HCN channels with subtype selectivity and high affinity.OBJECTIVES1)Explore the regulation of PIP2 on HCN channels.2)Investigate the effect of PI3K/PIP3,signaling pathways downstream of PIP2,on HCN channels.3)Discover a series of novel HCN channels inhibitors.METHODS1)Co-transfection HCN channels and PI5K recombinant plasmids into COS7 cells to investigate the regulation on HCN channels with the increasing of intracellular PIP2.2)Co-transfection HCN channels and M1 receptor recombinant plasmid into COS7 cells.20 ?M Oxo-M was used to activate the PLC signaling pathway to explore the effects of PIP2 down-regulation on HCN channels.3)Construction of stable HEK293 cells line with expressing of HCN4 channels and co-transfection PI3K in stable HEK293 cell line to investigate the regulation on HCN4 channels with over-expression of PI3K.4)A series of compounds were screened on HCN2 channels.Further chemical modi-fication was used to search novel inhibitors of HCN channels with highest affinity and subtype selectivity.RESULTS1)Over-expressed PI5K can specifically regulate HCN4 channel.2)Over-expressed M1 receptor to activate PLC signaling pathway can enchance currents of HCN channels slightly.3)Up-regulated PIP3 levels by over-expressed PI3K can increase the function of HCN4 channel.4)Compound 4a can inhibit HCN channels.Further chemical modification leads to the discovery of 4e,which possess the highest affinity towards HCN2 in our study.5)4e blocks the Ih in small DRG neurons and inhibits its action potential discharge frequency.CONCLUSIONOur results suggest that up-regulation of intracellular PIP2 levels by overex-pressed PI5K can specifically regulate the function of HCN4 channel,which is mainly reflected in affecting the voltage sensitivity,current amplitude and activation kinetics In addition,up-regulation of intracellular PIP3 levels though overpression of PI3K could also upregulate the function of HCN4 channel,mainly by increasing its current density and slowing down the activation speed dramatically.Moreover,our subject has discovered a series of inhibitors on HCN channels with novel structural frameworks,in which compound 4e preferentially blocked HCN2 channel with a lower IC50 value(3 ?M)compared with HCN1 and HCN4 channel.Further,our study show 4e blocked Ih and action potential firing property in small DRG neurons,which indicates its possible application in pain treatment.