The Study On The Interaction Of Ion Currents In The Two Delayed Rectifier Potassium Channels

Background and purpose:There are interactions between the different subunits of the same activatingdelayed rectifier potassium channel and different potassium channels.. Thisstudy is about interaction of different proportions between the subunits,comparing of the electrophysiological characteristics of KCNQ1/KCNE1currentand wild-type Ikscurrent, and comparing of the current characteristics ofcotransfected KCNQ1/KCNH2channel and wild-type KCNQ1channel andKCNH2channel, as well as the further revealing the incidence basis ofarrhythmia, and providing a theoretical basis for its clinical application.The First Part Beads Mark Method for the Determination of theKCNQ1/KCNE1Channel Current after the co-transfectionMethods:The KCNQ1/KCNE1channel expression system was made usingImmunomagnetic Beads positive mark method. The I-V curve ofKCNQ1/KCNE1current, the steady-state activation curve, the inactivation timeconstant and the difference with the WT-KCNQ1channel current were all usedto evaluate current characteristics of KCNQ1/KCNE1channel subunits underdifferent proportions.Results:This study got the cells positive rate of10¹5%with2ug plasmid and5ullipofectamine transfection reagents petri35mm dish in48-72h. RecordKCNQ1/KCNE1channel current, compare the peak current and tail current ofsubunits under the different proportions and when the proportion is up to1:3,the current reaches its peak; V_1/2and slope （k） is always larger than the valueof the WT-KCNQ1channel as the good fitting of the steady-state activationcurve with the Boltzmann equation; The deactivation time increase as the valueof deactivation time constant Tau1and Tau2are all large; the activation timeconstant Tau of KCNQ1/KCNE1（1:3） channel increase with the grow of the voltage.Conclusions:1. Beads mark method got the optimal transfection ratio:2ug plasmid and5ullipofectamine transfection reagents, the optimum ratio of plasmids betweenKCNQ1and KCNE1is1:3, the current of such combination is close to theclassic current Iks.2. The recorded peak current and the tail current of KCNQ1/KCNE1channel（α：β=1:3）are both larger than the current of wild-type KCNQ1channel,reflecting that protein expression quantity in KCNE1channel can increase theIkscurrent density.3. The gating dynamics characteristics of KCNQ1/KCNE1channel afterco-transfection is exactly opposite with the KCNQ1channel, but in accord withthe classic Iks, showing the slow activation, slow deactivation and nodeactivation current charateristcs.4. There is inseparable relation between the change of the current density andgating dynamics and the subunits interaction in KCNQ1/KCNE1channel.The Second Part Electrophysiological Characteristics of theKCNQ1/KCNH2Channel and its difference with KCNQ1channel KCNH2channelMethods:This study would reveal the interaction between different channels ofpotassium channels, and the mechanism of the causing of arrhythmias byrecording KCNQ1/KCNH2channel current through the method ofimmunomagnetic Beads positive mark, and comparing with separate transfectedKCNQ1channel and KCNH2channel current.Results:The peak current and tail current of KCNQ1/KCNH2channel under thefunction of the KCNQ1channel protein increase significantly. When thestimulus voltage was up to20mV, the characteristics of the inward rectifiercurrents were shown. The channel steady-state activation curve fit well with the Boltzmann equation as use the-40mV⁺60mV series pulse stimulation,showing obvious S-shaped curve and V_1/2=-0.92331±2.22338mV，k=9.58502±2.03137mV; the activation time constant is obviously larger thanthat of separate KCNQ1channel and KCNH2channel, but Tau value decreasedramatically when the voltage reach20mV, in accord with the I-V curve of itspeak current.Conclusion:1. the current density of co-transfected KCNQ1/KCNH2potassium channelcurrent is larger than that of the wild-type, but doesn’t change thecharacteristics of the inward rectifier. That is to say synergistic effect ofKCNQ1channel protein can significantly change the curve shape and peak ofthe KCNH2channel current.2. Steady-state activation curve fits with Boltzmann equation when stimulusvoltage is-40mV⁺60mV.The deactivation time constant and activation timeconstant of KCNQ1/KCNH2potassium channel is obviously larger than that ofseparate wild-type channel, the longer time, the current amplitude increasingcorrespondently, in line with its I-V curve.3. The dynamics change of co-transfected KCNQ1/KCNH2potassium channelindicates there is interaction between Iksand Ikrchannel of potassium channel.