Electrophysiological Characteristics Of The T618I Mutation HERG Potassium Channels And The Drug Reactivity By Beads Marked Method

This study is about the comparing of the electrophysiological characteristics ofwild type and T618I mutant hERG potassium channel, and the observing of dofetilidereactions of the T618I mutant hERG potassium channel, as well as the furtherrevealing the theoretical basis of short QT syndrome and its clinical application.The first part Beads mark method for the determination ofthe wild-type hERG potassium channel currentMethods: The hERG potassium channel expression system was made using Beadsmark method. The IV curve of the wild-type hERG potassium channel, thesteady-state activation curve, the steady-state inactivation curve, the inactivationtime constant and the action potential clamp were all used to evaluated the value ofthe system utilities.Results: This study got the cells positive rate of10~20%with2ug plasmid and5ullipofectamine transfection reagents petri35mm dish in48-72h. When the stimulusvoltage was up to20mV, the characteristics of the inward rectifier currents wereshown. The V1/2of steady-state activation curves was-12.2±0.629mV, and the slope(k) was11.8±0.587mV. The V1/2of steady-state inactivation curves was-11.9±2.75mV, and the slope (k) was19.0±1.75mV.The deactivation time constant increasedthe voltage10mV. Under the action potential clamp stimulation, the current reachedits peak when the voltage is-40mV.Conclusions:1. Beads mark method got the optimal transfection ratio:2ug plasmid and5ullipofectamine transfection reagents petri35mm dish in48-72h.2. The HERG potassium channel current that recorded was typical, and thesteady-state activation and inactivation curves were fit well with Boltzmann equation.3. The deactivation time constant and action potential I-V curves also confirmed thatthis method can record the typical hERG potassium channel current. The second part Electrophysiological characteristics of the T618Imutation hERG potassium channels and their drug reactivityMethods: This study would reveal the mechanism of the causing of arrhythmias bycomparing the wild-type hERG potassium channel current and the T618I’s andobserving dofetilide response of the T618I mutation hERG potassium channels.Results: When the stimulation voltage was up to60-70mV, the instability of T618Imutation hERG potassium current was appeared; then to80mV, the characteristics ofinward rectifier current was confirmed. T618I steady-state activation curve was fitwell with Boltzmann Equation under the potential of-50mV to+50mV(V1/2is0.6623±1.167mV, slope (k) is13.01938±1.208mV). The steady-state inactivationcurve was not fit well with BE. The asymmetric IV curve of the action potentialclamp was skew to the right.30nmol/L dofetilide can reduce the mutant channelcurrent, and put the stimulus voltage which leading the inward rectifier current,moving negative.Conclusions:1. The T618I mutation makes the hERG potassium current amplitude larger than thewild-type, and put the stimulus voltage which leading the inward rectifier current,moving positive.2. The steady state activation curve of T618I mutant hERG potassium channel is fitwell with Boltzmann Equation in the stimulating potential of-50mV to+50mVexcept the stimulus voltage exceeding50mV when the steady-state inactivation doesnot meet the Boltzmann Equation.3. The arrhythmogenic properties were shown through the application of three kindsof action potential clamp stimulus.4. Dofetilide not only reduce the current amplitude of the T618I mutant hERGpotassium channel, but also enable the stimulus voltage to lead the inward rectifiercurrent moving to the negative direction.