Study On The Opening Effect Of Compound QO-83 On KCNQ Channel

KCNQ?Kv7?channel is a voltage-dependent potassium ion channel.Currently,five subtypes are found.KCNQ1 is distributed in myocardium,KCNQ2 and KCNQ3 are mainly distributed in nervous system,KCNQ4 is distributed in inner ear hair cells and vascular smooth muscle,and KCNQ5 is distributed in nerve and skeletal muscle.Kv7.2/Kv7.3 or Kv7.3/Kv7.5channels formed by KCNQ2,KCNQ5 and KCNQ3 genes are the molecular basis of M current in nervous system and play an important role in regulating nerve excitability.QO-83 is a small molecular compound optimized by high-throughput screening.It has good regulatory activity on KCNQ channels and is expected to be developed into drugs for treating neurological diseases such as epilepsy and pain.Objective:To study the opening effect of QO-83 on various subtypes of Kv7 channel by electrophysiological patch clamp technique,and to explain its mechanism of action by combining point mutation and channel dynamics process.Methods:1 Electrophysiological patch clamp technique was used to observe the opening activity of QO-83?10?M?to different subtypes of Kv7 channel on expression cell lines.2 The whole cell current of Kv7 channel was recorded by patch clamp in the expression system.The effect of QO-83?0.1,1.0,3.0,10,30,100?M?with different concentration on the current was observed and the dose-effect curve was drawn.3 Electrophysiological patch clamp was used to record channel current,analyzed voltage-dependent characteristics,and observed the effect of QO-83?10?M?on channel activation curves.4 The whole cell current of Kv7 channel was recorded,the mechanical parameters of activation and deactivation were analyzed,and the effect of QO-83?10?M?on the dynamic parameters of Kv7 channel was observed.5 Point mutations KCNQ2 channel sites Kv7.2?W236L?,Kv7.2?Y284C?and Kv7.2?A306T?were used to study the effect of QO-83 on the sensitive site of Retigabine?RTG?.Results:1.Compound QO-83 had a significant opening effect on Kv7.2?Kv7.5channels?except Kv7.3 channel?.The current opening multiples of Kv7.2,Kv7.4,Kv7.2/Kv7.3,Kv7.3/Kv7.5 channels were 1.65±0.16 times,1.41±0.07 times,1.61±0.21 times and 1.27±0.13 times.2.QO-83 had no obvious opening effect on Kv7.1 and Kv7.3 channel at100?M concentration,and the EC₅₀ opening potential values for Kv7.2/Kv7.3and Kv7.4 channels were 1.9±0.8?M and 1.8±0.4?M respectively.3. Compound QO-83?10?M?could move the steady-state activation curves of Kv7.2,Kv7.2/Kv7.3,Kv7.4,Kv7.3/Kv7.5 channels towards hyper polarization,with?V_1/2 values of 19.3±3.6 m V,16.5±1.8 m V,13.0±2.3 m V,6.0±1.8 m V respectively,which were significantly different from those of the control group?P<0.05?.4. Compound QO-83 could significantly prolong the deactivation time of Kv7.2,Kv7.2/Kv7.3,Kv7.4,Kv7.3/Kv7.5 channels at a concentration of 10?M.The activation time constants(?_act,ms)of the channels analyzed at an activation voltage of-30m V were 331.4±49.2,368.9±56.4,368.6±55.4,325.0±29.8,respectively.There was no statistical difference compared with the?_deact value of the control group.The deactivation time constants(?_deact,ms)of the channels were 110.9±21.7,79.8±10.2,46.8±9.3,84.1±10.2respectively,which are statistically different?P<0.05?compared with the control group?_deact.5. Compared with RTG-sensitive Kv7.2?W236L?mutation sites,QO-83?10?M?also had no effect.For epilepsy-sensitive mutation sites Kv7.2?Y284C?and Kv7.2?A306T?,QO-83 and RTG were both effective.QO-83?10?M?could not only increase Kv7.2?Y284C?and Kv7.2?A306T?channel currents,but also shift the channel activation curve left by?V_1/2?m V?to 11.8±2.6 and 14.5±3.0 respectively,it had no obvious influence on the activation kinetic constant.The?_act values were 138.6±30.1 ms and 334.6±57.7 ms respectively,but the deactivation time values(?_deact)were 116.4±29.1 ms and 118.5±17.2 ms respectively?P<0.05?.Conclusion:Compound QO-83 showed obvious opening subtype selectivity,concentration dependence and voltage dependence on Kv7 channel.It was highly selective to Kv7.2 and Kv7.4,but had no obvious effects on Kv7.1 and Kv7.3.The mechanism of QO-83 opening channel was mainly to prolong the process of deactivation.Compared with RTG,QO-83 had a big difference that was no obvious selectivity for Kv7.3 subtype.