The Effect Of Compound C15as Potassium Channel Openner On KCNQ Currents

The KCNQ gene encodes a member of the Kv7family of K+channelsubtype. This family contains five members (KCNQ1-KCNQ5), among these,two channels, KCNQ2and KCNQ5, could co-express M-type currents (Im) inthe nervous system, which have characters as slow activation,nondeactiovation and voltage dependent. M channels as a low thresholdvoltage gated potassium play an important function in regulating nervousaction potential, the nerve cell excitability, rest memberane potential andsynaptic transmitter release etc. Regulating KCNQ potassium channel, it hasbeen as an key pathway to cure epilepsy, convalsions, pathological pain andsuch neurological diseases. Some regulater of KCNQ such as Diclofenac,Flupirtine, Both drugs have a potential enhance the KCNQ currents to curepain as a non opioid analgesic.Many KCNQ openers were reported in recent year. Selective neuronalKCNQ channel openers have been identified, these drugs already in humanuse, including diclofenac (a widely used anti-inflammatory agent), flupirtine(a non-opioid analgesic in approved use in Europe) and retigabine (which iscurrently in clinical trials). Additional potent and selective KCNQ channelopeners have recently.Compounds C15as a new chemical compound had a potential activity toopen KCNQ channels, discovered by high-throughput screening method. Theproject studied the detail, the mechanism and the characteristics about the newchemical compound C15, in regulation KCNQ channels. The aim is to providesome valuable experiment data for the next exploration.Aim: By means of whole cell patch clamp system, to record KCNQ andM currents in HEK293B cells as expression systems and DRG neurons asprimary culture cells By means of currents clamp technology, to observe the effect of compound C15on the action potential and analysis the excitabilitychanges.Methods:(1) Channel expression: cDNA coding KCNQ2,KCNQ3were insertedinto the pcDNA3.0plasmid vector, cDNA coding GFP was inserted intopEGFP-N1plasmid vector and transformed in TOP10competent germ. Afterextract and amplification, plasmids were detected with agarose gelelectrophoresis and ultraviolet light spectrophotometer. The KCNQ2,KCNQ3and GFP plasmids were co-expressed in HEK293cells, to observe the greenfluorescence cells with the help of fluorescence microscope which set up themotivation wave450~490nm.(2) Potassium channel currents recording: with Axon700B system,KCNQ currents was produced from HEK293cells, co-transfected KCNQ2/3plasmids and perforated whole-cell patch clamp by Amphotericin B. We alsoobserved the regulation of compound C15for M-currents in DRG neurons.(3) Observe the effect of C15for neuronal M current in primary culturesof DRG neurons, by the whole-cell patch-clamp technique.(4) Recording membrane potential: using Axon700B system, werecorded the action potential of DRG neurons by current clamp method, andobserve the effects of C15and XE991on neurons excitability.Results:(1) Amplification and Extract of plasmid cDNAs: Bands of pcDNA3.0plasmids (5.4Kbp) cloned KCNQ and pEGFN-1plasmids (4.8Kbp) showednearby5000bp in the electrophoresis. ND-1000ultraviolet lightspectrophotometer measured the density of plasmid cDNAs: KCNQconcentrations were between200~500ng/μL, GFPwas between200-500ng/μL;OD260/OD280values was between1.8~1.9.(2) The compound C15enhanced the KCNQ2/3currents in a way ofconcentration dependent. The compound C15(10μmol) increased theactivation and deactivation currents significantly. At the concentration rangefrom0.01μmol to60μmol, we use the logistic equation to fit the concentration-reaction curve, the EC50value0.59±0.03μmol (n=8).(3) The compounds C15shift the KCNQ2/3channels activation curve. Ifwe normalized the tail currents at the different voltage, could calculate the I-Vactivation curve. The compound of C15, under the different concentration0.04,0.2,1,5,20μmol, could gradually move the channel activation curve tohyperpolarized direction. The parameter of V1/2value changed, UsingBoltzmann equation fitted the curve, got the EC50value0.8±0.1μmol.(4) The compound C15changed the KCNQ2/3channels kineticparameter: Tau value, a parameter to evaluate the channel kineticcharacteristics, got from a single exponential equation which used to fitactivation and deactivation curves. The compound C15obviously prolongedthe KCNQ channel activation and deactivation course (P<0.05, n=5).(5) The selectivity of compound C15on the different KCNQ subtypechannels. Compound C15also enhanced KCNQ1, KCNQ2, KCNQ4, KCNQ5,KCNQ2/3channels currents and make the activation curve shift to thehyperpolarization (leftward shift). The subtype of KCNQ3had no obviousreaction to the compound C15(1μmol) compared with the KCNQ2/3channel.The main effect of C15on the all kinds of KCNQ subtype channel wasprolongation the deactivation course more distinguish than activation course.(6) The compound of C15also effect the mutation of KCNQ channel, theeffect of C15(1μmol) on KCNQ2(R207W) and KCNQ2(A306T) wasobvious, but have no much effect on KCNQ (Y284C) channel. while KCNQ2(Y284C) had scarcely reaction to the compound C15.(7) The effect of C15on M current recording from rat dorsal rootganglion. Recording M currents in rat DRG neurons. Compared with RTG, theeffect of C15have no significant difference (P>0.05, n=8). The compoundC15(10μmol) significantly increase M currents and cause the channel currentincreased21%(n=8). The compound C15make the resting membranepotential in the dorsal root neurons, it can be eliminate by XE991. On theother hand, the compound C15reduced the excitability of neuronssignificantly, and had an action to eliminate the clusters action potential. Conclusion:(1) The compound C15rapidly enhance KCNQ2/3channelcurrent in a way of concentration-dependent, the EC50is0.59±0.03μmol, itcould leftward shifted the KCNQ2/3channel activation I-V curve, and slowedchannel activation and deactivation kinetics.(2) C15active KCNQ1, KCNQ2,KCNQ4and KCNQ5, had little effect on KCNQ3channel. While have mucheffect on KCNQ2/3. C15also slowed channel deactivation and activationkinetics of KCNQ channels.(3) The compound C15increase native neuronsM current and cause marked membrane hyperpolarization of DRG neurons,reduce depression of evoked action potentials.