Study Of Electropharmacological Properties Of HNav1.5Sodium Channels

Part oneElectropharmacological properties of different stereoisomeric forms of glycyrrhetinic acid on wild type hNav1.5channelsObjective:The aim of the study was to investigate electropharmacological properties of18β-glycyrrhetinic acid (18β-GA) and18a-glycyrrhetinic acid (18α-GA) on wild type hNav1.5in Xenopus oocytes to assess the difference of action of18β-GA and18a-GA on wild type hNavl.5.Methods:Vector plasmid pTSV40containing wild type hNavl.5cDNA fragment was prepared to hNavl.5mRNA in intro transcription and injected in Xenopus oocytes. The standard two microelectrode voltage clamp technique was employed to record sodium current.Results:In wild type hNav1.5channels,100μM18a-GA has no significant effect on hNav1.5currents. However,18β-GA produced voltage-and concentration-dependent and infrequency-dependent inhibition wild type hNav1.5channels. The peak of I-V curve was shifted to more positive. And the peak Iua at-20mV was reduced from-5.93±0.46μA to-3.41±0.42μA (P<0.001, n=6) after application of30μM18β-GA and was almost completely recovery after washout (-5.66±0.53μA, P>0.05);18β-GA significant shifted the V1/2of activation curves in the positive direction and the steady-state inactivation curves in the negative direction and these shifts were completely recovery after washout. The slope factors were significantly increased in the presence of18β-GA.The mean values of half-activation (V1/2) were-33.84±0.74mV and-25.06±0.76mV before and after the application of30μM18β-GA, respectively (P<0.001,n=6);The half-inactivation potentials (V1/2) were-81.82±0.16mV and-88.09±0.12mV for control and application of30μM18(3-GA, respectively (P<0.001, n=6);18β-GA at30μM caused significant slowing of the recovery of peak INa-The time constants for both the fast and the slow recovery component (tf and ts) was significantly increased compared with control (if=11.79±1.00ms and22.05±3.03ms, respectively, P<0.01; and ts=261.72±10.60ms and342.71±14.74ms, respectively, P<0.05). With the first pulse of the train after10minutes18β-GA exposure, peak INa amplitude was already reduced to of control at three stimulating frequencies, and only a little additional block occurred at4Hz rate-dependent block of wild type Navl.5. On the contrary, lidocaine caused a significant frequent-dependent reduction (P<0.01, n=5).18β-GA block of peak INa in concentration-dependent manner after the successive application of1,10,30and100μM18β-GA and exhibited IC50values of39.79±3.29μM.Conclusion:18β-GA produced voltage-and concentration-dependent and infrequency-dependent inhibition wild type hNavl.5channels through change the kinesics of wild type hNavl.5channels.100μM18a-GA has no effects on the function of wild type hNav1.5channels. Part twoElectropharmacological properties of18β-glycyrrhetinic acid on mutant hNavl.5channelsObjective:The aim of the study was to investigate electropharmacological properties of18α-GA on mutant hNav1.5in Xenopus oocytes to assess the action of18β-GA on mutant hNav1.5.Methods:Vector plasmid pTSV40containing mutant hNav1.5-AKPQ cDNA fragment was prepared to mutant hNavl.5-AKPQ mRNA in intro transcription and injected in Xenopus oocytes. The standard two microelectrode voltage clamp technique was employed to record sodium current.Results:In mutant hNavl.5-AKPQ channels, application of18β-GA decreased peak and late INa of hNav1.5-AKPQ channels.18P-GA also produced voltage-and concentration-dependent and infrequency-dependent inhibition mutant hNavl.5-AKPQ channels. Peak INa of AKPQ channels was reduced by18P-GA at100μM at every potential in the range from-40mV to+20mV. The mean peak INa of hNavl.5-AKPQ channels at-20mV were-10.55±0.64μA and-6.10±0.65μA for control and18β-GA at100μM (n=6, P<0.001). The late INa of hNav1.5-△KPQ channels was also reduced by18β-GA. The mean late INa of hNavl.5-AKPQ channels were-0.39±0.12μA and-0.067±0.0087μA for control and18β-GA at100μM (n=10, P<0.05). Steady-state activation curve were significant shifted to positive direction by18β-GA at100μM. By contrast, steady-state inactivation of peak INa of hNavl.5-△KPQ channels was shifted significantly to the negative direction.18p-GA at100μM also significantly slowed of the recovery of peak INa of hNavl.5-AKPQ channels. The time constants for both the fast and the slow recovery component (rf and rs) was significantly increased compared with control (tf=5.77±0.44ms and7.25±0.82ms, respectively, P<0.05; and ts=201.20±16.54ms and271.98±13.89ms, respectively, P<0.01). Similar to wild type hNavl.5, with the first pulse of the train after10minutes18β-GA exposure, peak INa amplitude was already reduced to of control at three stimulating frequencies, and only a little additional block occurred at4Hz rate-dependent block. But late INa exhibited a significant rate-dependent reduction.18P-GA block of peak and late INa carried by hNavl.5-ΔKPQ in concentration-dependent manner after the successive application of1,10,30and100μM18β-GA and exhibited IC50values of100.39±4.59μM and37.19±5.83μM, respectively.Conclusion:18(3-GA produced voltage-and concentration-dependent and infrequency-dependent inhibition mutant hNavl.5-△KPQ peak INa through change the kinesics of mutant hNavl.5-△KPQ channels.18β-GA exhibited greater degrees of tonic inhibition of late INa compared with peak INa for hNavl.5-△KPQ channel and exhibited a significant rate-dependent reduction. Part threeElectropharmacological properties of18β-glycyrrhetinic acid on mutant HERG and hKvl.5channelsObjective:Blockage of HERG and hKvl.5is believed to cause long QT sysdrome, which can induce early after-depolarization and a Torsades de-Pointes-type of ventricular arrhythmia. The aim of the study was to investigate electropharmacological properties of18β-GA on HERG and hKv1.5channels in Xenopus oocytes to assess the risk of18β-GA on ventricular arrhythmia.Methods:Vector plasmid psP64and pCI-neo containing HERG and Kvl.5cDNA fragment was prepared to HERG and Kv1.5mRNA in intro transcription and injected in Xenopus oocytes, respectively. The standard two microelectrode voltage clamp technique was employed to record sodium current.Results:In HERG and Kv1.5channels, application of100μM18β-GA had no inhibitory effects on either HERG or Kvl.5channels (P>0.05, n=6). Conclusion:18β-GA had no significant change the kinesics of HERG and Kvl.5channels and the risk of induce early after-depolarization and a Torsades de-Pointes-type of ventricular arrhythmia through cause long QT sysdrome.