Study Of Molecular Determinants And Function Of HERG Channels Interacting With Agents In Acquired Long QT Syndrome

Part one The persistent expression of HERG channel in Xenopus Oocytes, the alteration of current and Analysis of HERG Channel KineticsObjective: To explore a method of the stable and persistent expression of HERG (human ether-à-go-go–related gene) channels in Xenopus oocytes, investigate the alteration of rest membrane potential of oocytes and electrophysiological properties of expressed channel in different culture duration,and to investigate the methods and mechanisms of analysis of channel kinetics parameters of HERG channels, in the process of electrophysiological recording.Methods: HERG mRNA for injection was prepared with in intro transcription using vector plasmid pSP64 containing HERG cDNA fragment. Expressed HERG current was recorded using standard two-microelectrode voltage-clamp technique and channel kinetics parameters were analyzed through compiling different pulse protocol and recording current.Results: (1) Functional channels, with electrophysiological properties consistent with those of HERG channels were persistently expressed in oocytes membrane with this method. Furthermore, channel current could be recorded stably in 10-15 days. (2) The negative value of rest membrane potential increased gradually in the 3, 6, and 9 days of culture, and then decreased in the 12 days. The potential of peak value of inward rectification shifted gradually to the positive direction in 3, 6 and 9 days, and recovered in 12 days. Half-maximal activation potential (V1/2) of meteorological expressed current shifted gradually to the negative direction in 3, 6 and 9 days of culture and then recovered in 12 days, the tendency of change was coincident with that of membrane rest potential. (3)HERG channels were activated with depolarized pulse and expressed an inward-rectified propriety attributing to rapid inactivation. The activation curve was obtained through fitting the depolarized potential and following peak amplitude of tail current, while parameters of time-dependent activation was got through fitting different depolarized duration and according peak amplitude of tail current. (4) The I-V relationship still exhibit marked inward rectification. Tail current decay traces were fitted with a bi-exponential function to determine the time constants of the fast and slow components of current decay. (5) The inactivation of HERG channels is voltage-dependent. The inactivation process was isolated with two different three-pulse protocols, with which the inactivation curve and nearly linear I-V relationship were obtained, respectively.Conclusion: The investigation provides a method of persistent expression of HERG channel in Xenopus oocytes and offer evidences for the difference of electrophysiological experimental data of studies of molecular site and drugs effect of HERG channel in different experimental conditions. Through the kinetics properties of HERG channels were complicated, the channels kinetics could be indirectly analyzed through designed different pulse protocol, which provided foundation for Alanine-scanning mutagenesis and agents'action investigation.Part two Astemizole preferentially blocks open HERG channels involving in binding to the S6 residue Y652 and F656Objectives: Second-generation antihistamines astemizole-induced block of cardiac HERG channel causes acquired long QT syndrome. We characterized the complete biophysical properties and the molecular determinants of HERG block by astemizole.Methods: Channels were expressed in Xenopus oocytes, and currents were measured using two-microelectrode voltage-clamp technique. Results: Astemizole blocked WT HERG current in voltage- and concentration-dependent manner. Astemizole showed minimal tonic block of HERG current evaluated by the envelope of tails test. Characteristics of block were consistent with an open channel blockade. The time constants of slow component of HERG deactivation were significantly increased by astemizole in concentration-dependent manner. The V1/2 values for steady-state inactivation and I-V relationships of fully activated current and steady-state current of HERG channel were not significantly altered by astemizole. While time course of channel inactivation was accelerated by astemizole in voltage-dependent manner. The S6 mutation, Y652A and F656A, significantly attenuated the blockade by astemizole. The IC50 value was increased by 11-fold for Y652A and 404-fold for F656A compared with WT current blockade.Conclusions: Astemizole preferentially blocks open HERG channels. Tyr-652 and Phe-656 are two critical residues in the astemizole-binding site, interacting with the side chain attaching to the tertiary amine within the molecular of antihistamines.Part three Biophysical properties and molecular determinants of wild type and mutant human ether- a` -go-go-related gene channel blockade by haloperidolObjectives:Haloperidol, an antipsychotic agent, prolonged the QT interval of ECG and caused lethal arrhythmia at therapeutic concentration through blocking human ether- a` -go-go-related gene (HERG) channel. We investigated the biophysical properties of wild type (WT) and mutant Y652A HERG channel blockade by haloperidol and characterized the molecular determinants of HERG blockade by it.Methods:HERG channels were expressed in Xenopus oocytes, and currents were measured using the two-microelectrode voltage-clamp technique.Results: Haloperidol blocked WT HERG current in concentration- and weak voltage-dependence manner and had no significant influence on activation curve of HERG current. Haloperidol showed tonic block of HERG current evaluated by envelope of tails test and inactivation curve was shifted to negative direction. While mutation of Y652A HERG had similar effect on HERG channel kinetic compared with that of WT channels. The S6 mutation, Y652A and F656A, significantly attenuated the potency of channel blockade by haloperidol. The IC50 value was increased by 21-fold for Y652A and 161-fold for F656A.Conclusion: Mutant channels of Y652A do not significantly alter biophysical properties of HERG channel blockade by haloperidol. Y652 and F656 are two critical molecular determinants for block of HERG channel by haloperidol.Part four The anti-psychotics thioridazine blocks HERG potassium channels involving closed and open states via binding to the S6 residue F656Objectives:Antipsychotics thioridazine was reported to lengthen repolarization of cardiac ventricular myocytes and QT interval of ECG by blocking HERG channel. The objectives of this study were (i) to characterize any inhibitory action on HERG of thioridazine, (ii) to then determine whether thioridazine share consistent molecular determinants of HERG blockade as other reported drugs.Methods: Heterologous HERG potassium current expressed in Xenopus oocytes was recorded using two-microelectrode voltage-clamp technique at room temperature.Results: Thioridazine blocked wild-type HERG current in concentration-dependent manner with an IC50 7.3μM. Thioridazine produced a tonic block on HERG, with a significant time-dependent decrease in HERG current at the initial depolarization duration and voltage-independent blockade at negative to +40mV depolarization. The inhibitory effect was attenuated at membrane potentials greater than 40 mV, which indicated that channel inactivation prevented blockade by thioridazine. Therefore, thioridazine preferentially blocks closed (resting) state channel, and also inhibits open-state channel rather than inactivated HERG channels. The fast and slow time constants of deactivation were accelerated, lack of "foot in the door" effect. The inactivation curve was shifted to the negative direction and inactivation rate was increased. The S6 mutation Y652A and F656A produced～4-fold and～25-fold increases in IC50 for HERG current blockade, respectively.Conclusions: S6 residue F656 is an important determinant of thioridazine binding though its closed-state blockade. Thioridazine is therefore quite distinct in its blockade mechanism from previously studied open-state blockade agents and perhaps similar to the inhibitory mechanism of the preferential closed channel blocker BeKm-1 on HERG channels.