| M channels are low-threshold, depolarization-activated andvoltage-dependent potassium channels that were first described by Brown andAdams in1980s. M channels were found expressed in many type of neuronsincluding sympathetic neurons, dorsal root ganglion neurons and centralneurons. It is now well established that the homomeric and heteromeric Kv7family channels (Kv7.2，Kv7.3and Kv7.5) underlies the molecular basis ofM currents. M channel plays a key role in controlling the excitability ofneurons. Mutations in Kv7.2and Kv7.3channel gene lead to a form ofneonatal epilepsy in humans termed BFNC (Benign Familial NeonatalConvulsions). Recently, M channels are also found in sensory neuronsinvolved in pain transmission. Thus, Kv7/M channel is an important target forstudying to develop modulators for the treatment of epilepsy and pain.The Kv7/M channel as molecular target for the novel anti-epileptic drughas become hot spot in research, and has already made great progress.Retigabine is the representative novel anti-epileptic drug. Retigabine has beenshown to have broad spectrum of anticonvulsant properties in animal tests andclinical trails, and has been shown to be efficacious and safe in patients withrefractory epilepsy. Marketing of Retigabine has been approved by the drugadministratives in the United States and Europe. Additionally, retigabine alsoshows analgesic activity, especially in animal models of chronic inflammatoryand neuropathic pain.Chloroprocaine is a new generation ester local anesthetic, having awith fast onset and, producing excellent sensory block actions in clinicaluse. Accidental intravenous injection, overdosage or rapid systemic uptake of local anesthetics may result in severe systemic toxic effects. The earliestsymptoms of toxicity from chloroprocaine are excitation of the central nervoussystem, including seizure induction. The molecular mechanisms underlyinglocal anesthetics-induced seizure are complicated. The toxicity was seriouscomplication of local anesthetic overdose. The mechanism underlying thetoxicity of local anesthetic overdose and the prevention were always, which isa focus of attention.In the first part of the study, we characterized the effects of a newKCNQ/M channel opener, QO-58on the currents of KCNQ channels. QO-58is the lead optimized compound identified in our high-throughput screening ofour synthesized series of pyrazolo[1,5-a]pyrimidin-7(4H)-one (PPO)compounds, using atomic absorption Rb+efflux assay. Among the PPOs wesynthesized, the compound QO-58showed strong activity of KCNQ/Mchannel activation. In the second part of the study, we tested nearly20localanesthetic drugs for their effects on the currents of Kv7.2/Kv7.3channel. Wefound the new generation local anesthetic chloroprocaine inhibitedKv7.2/Kv7.3channel. In the third part of the study, we found that a novelcompound SCR2682potently enhanced Kv7.2/Kv7.3channel currents. Alsothe relation of the effects of above compounds on epilepsy to their modulationof Kv7/M currents was also studied.Part1The effects of compound QO-58on Kv7potassium currents,neuronal excitability and pain behaviorObjective: We aimed to study the effects of pyrazolo[1,5-a]pyrimidin-7(4H)-one compound QO-58on Kv7/M channel currents andthe underlying mechanisms; to study the effects of QO-58on DRG neuronexcitability; to study the effects of QO-58on pain behavior of rats.Methods: Amphoterincin B perforated patch clamp technique was usedto study the effect of QO-58on Kv7/M channel currents expressed in HEKand CHO cells. Current clamp recording mode was used to monitor the effectsof drugs on membrane potentials and excitability of DRG neurons. Chronicconstriction injury of the sciatic nerve model was used to study the effect of QO-58on neuropathic pain.Results:(1) Compound QO-58activated Kv7.2/Kv7.3currents recordedat-40mV concentration-dependently with a EC50value of2.3±0.8μM and aHill coefficient of0.6±0.1. QO-58(100μM) induced a maximal6.15±0.76folds increase of the current amplitude recorded at-40mV. Compound QO-58concentration-dependently leftward shifted the Kv7.2/Kv7.3activation curve,with half-maximal channel activation (V1/2) occurring at-13.2±1.1mV,-23.9±1.5mV,-29.9±1.5mV,-38.9±1.4mV,-53.5±2.0mV and-58.2±1.7mVin the presence of0,0.1,0.3,1,3and10μM QO-58, respectively. QO-58concentration dependently shifted voltage-dependent activation ofKv7.2/Kv7.3channel, with a EC50value of1.2±0.2μM and a Hill coefficientof1.2±0.3. Application of QO-58significantly slowed channel activation anddeactivation kinetics. The time constant of current activation at-40mV was76.1±2.5ms for the control and179.5±25.8ms for QO-58(10μM). And forthe current deactivation at-120mV, the time constant was28.3±6.3ms forthe control and278.5±57ms for QO-58(10μM).(2) QO-58(10μM)increased significantly the Kv7.1, Kv7.2, Kv7.4and Kv7.3/Kv7.5currents by2.3±0.5,4.0±0.6,2.1±0.4, and1.1±0.6folds, respectively, but only slightincreased Kv7.3currents0.3±0.1folds. QO-58concentration-dependentlyincreased homomeric Kv7.1-4and heteromeric Kv7.3/Kv7.5channel currentsrecorded at-40mV; the EC50s value for QO-58were7.0±1.0μM,1.3±1.0μM,0.6±0.1μM,5.2±2.2μM, for Kv7.1, Kv7.2, Kv7.4and Kv7.3/Kv7.5channel, respectively. QO-58(10μM) produced a substantial leftward shift ofV1/2of Kv7.1, Kv7.2, Kv7.4and Kv7.3/Kv7.5currents by21.7±1.1mV,56.8±5.4mV,58.7±2.9mV and47.4±2.8mV, respectively; on the other hand,the V1/2of Kv7.3was shifted to the right only by2.7±0.1mV. QO-58significantly slowed the activation kinetics of Kv7.4and Kv7.3/Kv7.5currents,and slowed the deactivation kinetics of homomeric Kv7.1-Kv7.4andheteromeric Kv7.3/Kv7.5currents. QO-58(10μM) increased the deactivationtime constant of Kv7.2from15±1.5ms to96.4±12.5ms, and greatly increased the deactivation time constant of Kv7.4from8.7±0.2ms to316.6±125.8ms.(3)QO-58(10μM) markedly enhanced the Kv7.2(W236L) currents(the currents were increased by1.6±0.4folds at-50mV) and caused aleft-shift of the activation curve with V1/2changed from-33.3±1.7mV to-92.8±2.7mV. The V1/2of mutant Kv7.2(VVY224,225,226AIC (Kv7.2(AIC)) channel was-81.0±2.2mV, which was changed to-88.0±2.2mV and-95.5±1.7mV in the presence of QO-58and retigabine, respectively. QO-58changed V1/2of (A306T) from-46.3±1.2mV to-74.8±2.8mV and changedV1/2of Kv7.2(L275A) from-30.2±1.9mV to-67.5±3.5mV.(4) The effectsof QO-58on Kv7.1/KCNE1currents were also studied. QO-58increasedKv7.1/KCNE1currents recorded at0mV with a EC50of7.7±1.9μM and aHill coefficient of1.1±0.3. QO-58(10μM) only modestly increased the Ikscurrents recorded from guinea pig cardiomyocytes (14.3±1.8%increase at0mV). QO-58(100μM) had no effect on duration of the action potentialrecorded from guinea pig papillary muscles.(5) Both QO-58and retigabineincreased M currents recorded from DRG neurons by approximately25%at10μM. QO-58also induced significant hyperpolarization of the restingmembrane potential (RMP) of DRG neurons (RMP was hyperpolarized from-59.7±1.2mV to-78.1±2.3mV). The perfusion of QO-58immediatelyabolished the repetitive firing. DRG neurons (6) QO-58(50mg/kg injectedintraperitoneally) significantly increased the threshold of the mechanical andthermal stimulus1-19th days after the operation of constriction injury of thesciatic nerve.Conclusion:1. QO-58rapidly and reversibly increased Kv7.2/Kv7.3channel currents. Compound QO-58concentration-dependently activatedKv7.2/Kv7.3currents recorded at-40mV with a EC50of2.3±0.8μM. QO-58concentration-dependently leftward shifted the Kv7.2/Kv7.3activation curveand slowed channel activation and deactivation kinetics.2. QO-58concentration-dependently activated Kv7.1, Kv7.2, Kv7.4and Kv7.3/Kv7.5with more selective effect on Kv7.2and Kv7.4, but had little effect on Kv7.3channel. QO-58significantly slowed channel activation and deactivation kinetics of Kv7channels.3. Val224Val225Tyr226, Arg306and Leu275inKv7.2may be involved in QO-58activation of Kv7channels.4. QO-58significantly enhanced Kv7.1/KCNE1channel currents. QO-58had negligiblyeffects on Ikscurrents of guinea pig ventricular myocytes and had no effect onthe action potential of guinea pig papillary muscle.5. QO-58enhanced nativeneuronal M currents and led to marked membrane hyperpolarization of ratDRG neurons, resulting depression of evoked action potentials.6. QO-58alsoelevated the pain threshold of in chronic constriction injury of the sciatic nervemodel. The results indicated QO-58is a potent Kv7modulator and can bedeveloped further to treat disease related with neuronal hyperexcitability.Part2The modulation of Kv7/M channel currents by local anestheticchloroprocaine and the relationship of this modulation with thelocal anesthetic-induced seizuresObjective: We aimed to study the effects of local anestheticchloroprocaine on Kv7/M channel currents and the relationship of thismodulation with the local anesthetic-induced seizures.Methods: Amphoterincin B perforated patch clamp technique was usedto study the effect of chloprocaine on Kv7/M channel currents expressed inHEK cells. Current clamp recording mode was used to monitor the effects ofchloprocaine on membrane potentials and excitability of DRG neurons. Thebehavior study for chloroprocaine-induced seizures was also performed.Results:(1) Chloroprocaine concentration-dependently inhibitedKv7.2/Kv7.3currents.(2) Chloroprocaine (10mM) shifted thevoltage-dependent activation of Kv7.2/Kv7.3, with V1/2changed from-26.8±1.7mV to-16.1±1.3mV. Chloroprocaine (10mM) significantlyslowed channel activation and accelerated deactivation kinetics. The timeconstant for the current activation at0mV was26.9±3.9ms for thecontrol and36.4±4.1ms for chloroprocaine; and the deactivation timeconstant was71.3±24.7ms for the control and65.0±15.0ms forchloroprocaine.(3) Chloroprocaine (30mM) inhibited the currents ofKv7.2, Kv7.3and Kv7.2/Kv7.3channel recorded at+40mV by23.2± 5.2%,66.2±3.9%and34.8±1.1%, respectively. Chloroprocaine (30mM) also shifted the voltage-depndent activation of Kv7.2, Kv7.3andKv7.2/Kv7.3. For this, V1/2of Kv7.2was changed from-25.3±2.7mVto-15.4±2.9mV; V1/2of Kv7.3was changed from-29.4±1.0mV to-17.8±1.2mV; V1/2of Kv7.2/7.3was changed from-28.4±1.7mV to-6.2±1.8mV.(4) Chloroprocaine (30mM) depolarized the restingmembrane potential (RMP) of DRG neurons, RMP was changed from-64.6±0.1mV to-48.1±0.1mV, which was reversed by retigabine, and RMP wasreversed to-66.2±0.3mV.(5) Retigabine (20mg/kg injectedintraperitoneally) reduced toxic effects of chloroprocaine, TD50ofchloroprocaine was increased from166.1mg/kg to212.4mg/kg and LD50of chloroprocaine was increased from317.5mg/kg to433.3mg/kg byretigabine; similarly TD50of bupivacaine (another local anesthetic) wasincreased from40.8mg/kg to51.5mg/kg and the LD50was increasedfrom58.5mg/kg to147.3mg/kg by retigabine.Conclusion:1. The local anesthetic chloroprocaine inhibitedKv7.2/Kv7.3currents amplitudes, shifted the voltage-dependent activationcurves to more positive potentials, and slowed the channel activation andaccelerated deactivation kinetics.2. Chloroprocaine showed more selectiveinhibitory effect on Kv7.3and Kv7.2/Kv7.3than on Kv7.2channel.3.Retigabine reversed the inhibition of M currents and abolished the membranedepolarization of the DRG neuron induced by chloroprocaine.4. Retigabinereduced toxic effects of chloroprocaine and bupivacaine in in vivo studies.Part3The effects of compound SCR2682on Kv7potassium channelsand seizure in mouse modelObjective: We aimed to study the effects of SCR2682on Kv7/M channeland seizure incidence in a mouse model.Methods: Amphoterincin B perforated patch clamp technique was usedto study the effect of SCR2682on Kv7/M channel currents expressed in HEKand CHO cells. Current clamp recording mode was used to monitor the effectsof SCR2682on membrane potentials and excitability of DRG neurons. Maximal electroshock assay model was used to study the effect of SCR2682on the seizure incidence in the mouse model.Results:(1) Compound SCR2682concentration-dependently activatedKv7.2/Kv7.3currents recorded at-40mV with a EC50of9.8±0.4nM and aHill coefficient of0.6±0.2. SCR2682(100nM) shifted V1/2of Kv7.2/Kv7.3from3.1±3.0mV to-43.3±1.6mV. SCR2682(100nM) significantlyaccelerated channel activation and deactivation kinetics. The timeconstants for the current activation at-40mV was increased by SCR2682from a single exponential of137.5±15.6ms to two exponentials of128.5±20.0ms and510.1±50.3ms; and the deactivation time constants at-120mV was increased from21.8±4.3ms to147.3±15.3ms.(2) SCR2682（100nM）enhanced the currents of Kv7.2, Kv7.3, Kv7.4and Kv7.3/Kv7.5channel by4.7±0.4,1.7±0.03,2.9±0.3and1.9±0.1folds, respectively.SCR2682concentration dependently increased Kv7.2, Kv7.3, Kv7.4andKv7.3/Kv7.5channel currents, with EC50sof26.3±1.3nM,11.2±2.3nM,28.8±8.7nM and11.1±1.0nM, respectively. SCR2682(100nM) shifted theV1/2of Kv7.2, Kv7.3, Kv7.4and Kv7.3/Kv7.5channel from-16.7±1.2mV to-57.7±1.4mV，from-4.6±3.0mV to-48.1±2.5mV，from-0.1±2.5mV to-21.3±2.8mV, and from-13.5±3.5mV to-47.9±3.7mV, respectively.(3)The V1/2of mutant Kv7.2(W236L) channel were not affected by SCR2682(-22.7±3.0mV vs-25.4±4.2mV in the absence and presence of SCR2682,respectivel) but was significantly affected QO-58(-82.9±2.0mV in thepresence of QO-58(10μM)).(4) Both SCR2682(100nM) and retigabine (10μM) increased M currents in DRG neurons with similar potency. SCR2682also induced significant hyperpolarization of the resting membrane potential(RMP) of DRG neurons, the RMP was changed from-56.7±2.8mV to-66.4±3.7mV. SCR2682abolished the repetitive firing of DRG neurons.(5)SCR2682dose-dependently decreased the seizure incidence of mouse inducedby maximal electroshock, with a ED50of1.64±0.3mg/kg.Conclusion:1. SCR2682increased current amplitudes, hyperpolarizedthe voltage-dependent activation curves, and slowed the channel deactivation kinetics of Kv7.2, Kv7.3, Kv7.4and Kv7.2/Kv7.3.2. SCR2682activated Kv7channel with similar mechanism as retigabine.3. SCR2682enhanced M currents and hyperpolarized the membrane potentials of rat DRGneurons, and depressed evoked action potentials.5. SCR2682reduced theincidence of seizure in mouse model and showed the potential for thetreatment of seizures.Summary: QO-58concentration-dependently increased Kv7.2/Kv7.3channel currents，leftward shifted activation curve and slowed deactivationkinetics of Kv7.2/Kv7.3channels. QO-58showed more selective effect onKv7.2and Kv7.4, but had little effect on Kv7.3channel.Val224Val225Tyr226, Arg306and Leu275in Kv7.2may be involved inQO-58activation of Kv7channels. QO-58decreased the excitability of ratDRG neurons. QO-58also elevated the pain threshold of neuropathic pain inchronic constriction injury of the sciatic nerve model. The results indicatedQO-58is a potent Kv7modulator and can be developed further to treatneuropathic pain.The effects of chloroprocaine on Kv7.2/Kv7.3currents were manifestedby inhibiting current amplitudes, rightward shifting the voltage-dependentactivation curves, slowing channel activation and accelerating deactivationkinetics. Retigabine significantly reversed the inhibition of Kv7/M channelcurrents and abolished the depolarization of the DRG neuron membranepotential induced by chloroprocaine. Retigabine increased convulsionTD50and LD50induced by chloroprocaine overdose and showed potentialto prevent the toxicity induced by local anesthetic chloroprocaine overdose.The effects of SCR2682on Kv7.2/Kv7.3currents were manifested byconcentration-dependently increasing current amplitudes, leftward shifting thevoltage-dependent activation curves, slowing channel deactivation kinetics.SCR2682activated Kv7.2, Kv7.3, Kv7.4and Kv7.3/Kv7.5channel. Themechanism of SCR2682for Kv7channel activation was the same asretigabine. SCR2682reduced the incidence of seizure in mouse model and showed the potential for the treatment of seizures. |
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