Identification And Interaction Mechanisms Of Novel Inhibitors Acting On Potassium Channels

Potassium channels are the most diverse and widely distributed group of ion channels,which play a vital role in the maintenance of resting membrane potential,the formation of action potential,cell proliferation and apoptosis and other physiological and pathological activities.Therefore,it is of great significance to discover novel potassium channel modulators for the study of the structure and function of potassium channels and for the treatment of potassium channel related diseases.This work proposes to excavate novel types of potassium channel inhibitor from the defense peptide library and the drugs of potassium channel related diseases.Defensins are antimicrobial peptides widely distributed in different organisms.Our research team reported the first defensin hBD2 inhibitor of potassium channel in 2015.On this basis,we continued to carry out more extensive research on the potential interaction between human defensins and potassium channels.Based on the sequence alignment and spatial structure analysis,we selected human a defensins HNP1 and HD5 and tested their effects on potassium channels by patch clamp technique.The results showed that both HNP1 and HD5 could inhibit Kv1.3 channel and the corresponding IC50 values were 102.0 ± 30.3 nM and 2.2 ± 0.2?M,respectively.Besides Kvl.3 channel,HNP1 and HD5 showed no or less effects on Kv1.2,KCNQ1,IKCa and hERG channels at the concentration of 1 ?M.HNP1 not only inhibited Kv1.3 channel expressed on HEK293T cells,but also inhibited Kv1.3 channel and IL-2 secretion in human CD3+ T cell,indicating its new function in the immune system.Although HNP1 and HD5 shared similar three dimensional structures,they inhibited Kv 1.3 channel with different mechanisms.HNP1 could simultaneously bind to the S1-S2 linker and the pore region of Kv1.3 channel,whereas HD5 only bound to the pore region.Different from HNP1 shifting the conductance-voltage curve of Kvl.3 channel in the right direction by nearly 9.5 mV and increasing the activation time constant,HD5 did not induce a notable shift of the conductance-voltage curve of Kv1.3 channel.Together,these findings revealed that human a defensins are novel endogenous inhibitors of Kv1.3 channel and they interact with Kvl.3 channel through distinct mechanisms.Human defensins HNP1,HD5 and hBD2 selectively inhibited immune related Kvl.3 channel and show no or less effects on other potassium channels.To investigate whether it is the common feature for human defensins acting on potassium channels,we further tested the effect of human defensin hBD3 on Kvl.3 channel.It was found that hBD3 could also inhibit Kv1.3 channel with the IC50 value of 187.6 ± 25.7 nM.hBD3 and hBD2 interacted with Kv1.3 channel through different mechanisms.hBD2 not only bound to the pore region of Kv1.3 channel as a pore blocker,but also bound to channel S1-S2 linker as a gating modifier to affect the voltage sensor movement.While hBD3 only bound to the pore region and showed no effect on activation of Kv1.3 channel.Compared to hBD2 selectively inhibiting Kv1.3 channel,1 ?M hBD3 also inhibited 50.6 ± 3.6%,29.9 ± 1.2%and 33.6 ± 1.0%of Kv1.2,KCNQ1 and hERG channel currents,respectively.These results indicated the differential interaction mechanisms between human defensins and potassium channels are diverse.Methotrexate(MTX)is one of small molecule chemical drugs and is widely used in the treatment of rheumatoid arthritis,psoriasis,multiple sclerosis and other autoimmune diseases.The current studies showed Kvl.3 channel in lymphocyte is a new target for the treatment of autoimmune diseases.Based on the structure and property analysis,we speculated that MTX is a new inhibitor of Kvl.3 channel and the result showed that MTX could inhibit Kv 1.3 channel with the inhibition of 79.9 ± 1.5%at 1 pM and the corresponding IC50 value was 41.5 ± 24.9 nM.Furthermore,1 ?M MTX could also inhibited 32.6 ± 1.3%,25.6%± 2.2%of Kv 1.1 and Kv 1.2 channel currents respectively and showed no obvious effects on other potassium channels.The chimeric and mutagenesis experiments showed that the outer pore region of Kv1.3 channel was the binding site of MTX and the residues Asp371,Thr373 of turret and His399 above the selectivity filter played important roles in their interaction.Since Kvl.3 channel is closely related to autoimmune diseases.MTX may also achieve its therapeutic effect by inhibiting Kv1.3 channel.These findings revealed a new mechanism for MTX in the treatment of autoimmune diseases.In summary,we identified that human a defensing HNP1 and HD5 selectively inhibit Kvl.3 channel with the IC50 values of 102.0 ± 30.3 nM and 2.2 ± 0.2 ?M,respectively.We also found human P defensin hBD3 could inhibit Kvl.3 channel through the pore region.Unlike other potassium channel-acting defensins,1 ?M hBD3 could also inhibit other potassium channel currents.In addition,we also found that MTX,a drug for treatment of autoimmune diseases,could inhibit Kv1.3 channel with the IC50 value of 41.5 ± 24.9 nM.The chimeric substitution and mutagenesis experiments indicated that MTX bound to the extracellular pore region of Kv1.3 channel.These work indicated that potassium channels could be inhibited by more and more novel inhibitors with more interesting functions and prospects.